EP4058587A1 - Lentiviral vectors in hematopoietic stem cells to treat wiskott-aldrich syndrome (was) - Google Patents

Lentiviral vectors in hematopoietic stem cells to treat wiskott-aldrich syndrome (was)

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Publication number
EP4058587A1
EP4058587A1 EP20886855.4A EP20886855A EP4058587A1 EP 4058587 A1 EP4058587 A1 EP 4058587A1 EP 20886855 A EP20886855 A EP 20886855A EP 4058587 A1 EP4058587 A1 EP 4058587A1
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European Patent Office
Prior art keywords
seq
nucleic acid
vector
enhancer element
sequence
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EP20886855.4A
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German (de)
French (fr)
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EP4058587A4 (en
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Donald B. Kohn
Ryan L. WONG
Roger Paul Hollis
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University of California
University of California Berkeley
University of California San Diego UCSD
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University of California
University of California Berkeley
University of California San Diego UCSD
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Publication of EP4058587A1 publication Critical patent/EP4058587A1/en
Publication of EP4058587A4 publication Critical patent/EP4058587A4/en
Pending legal-status Critical Current

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
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    • A61P37/04—Immunostimulants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N15/867—Retroviral vectors
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE

Definitions

  • P220P_ST25.txt created on November 9, 2020 and having a size of 82.1 kb.
  • the contents of the text file are incorporated by reference herein in their entirety.
  • Wiskott-Aldrich Syndrome is an X-linked primary immune deficiency caused by mutations in the Wiskott-Aldrich Syndrome (WAS) gene.
  • WAS Wiskott-Aldrich Syndrome
  • Patients with WAS have severe defects in both adaptive and innate immunity and are highly susceptible to life- threatening viral and bacterial infections. Patients also suffer from extremely severe eczema, microthrombocytopenia and have a high risk of developing autoimmunity and cancer,
  • WAS occurs most often in males due to its X-linked recessive pattern of inheritance, affecting between 1 and 10 males per million.
  • the first signs are usually petechiae and bruising, resulting from a low platelet count (i.e, thrombocytopenia).
  • Spontaneous nose bleeds and bloody diarrhea are also common, and eczema typically develops within the first month of life.
  • Recurrent bacterial infections typically develop by three months.
  • the majority of children with WAS develop at least one autoimmune disorder, and cancers (mainly lymphoma and leukemia) develop in up to a third of patients.
  • Immunoglobulin M (IgM) levels are typically reduced, IgA and IgE are typically elevated, and IgG levels can be normal, reduced, or elevated.
  • WAS patients have abnormally small platelets (i.e. microthrombocytes) and -30% also have elevated eosinophil counts (i.e., eosinophilia).
  • Treatment for WAS typically involves prophylactic antibiotic and antiviral therapy to manage infections. Platelet transfusions and splenectomy are used to treat thrombocytopenia (low platelet counts). Additionally, blood transfusions may be required to treat anemia resulting from excessive bleeding and a protective helmet may be used to prevent brain hemorrhages which could result from head injury. Immunosuppressive treatment is utilized for autoimmune manifestations.
  • a potential curative therapy is an allogeneic hematopoietic stem cell transplantation from a HLA matched donor. However, this is not a viable option for many patients due to the unavailability of a suitable matched donor.
  • An alternative curative therapy is an autologous hematopoietic stem cell
  • HSC HSC transplantation with ex vivo gene therapy.
  • the patient acts as their own donor, eliminating the risk of immunological complications.
  • Successful implementation of this approach relies on the development of a lentiviral vector or CRISPR based therapy to introduce a functional copy of the gene of interest or to correct the pathogenic mutation in the patient’s HSCs.
  • Previous viral-based therapies utilized the CMMP-WAS g-retroviral vector.
  • Another therapy uses a safer SIN lentiviral vector driven by a 1.6kb promoter fragment of the endogenous WAS gene.
  • T, B, and NK cells were functional and normal immune cell counts were restored. Additionally, there was a decrease in severity and frequency of infections and severe eczema was resolved. However, platelet counts and mean platelet size remain under normal values. The patients remained microthrombocytopenic and retained a risk of severe bleeding episodes.
  • LVs novel lentiviral vector(s)
  • WAS Wiskott-Aldrich Syndrome
  • the vectors described herein show better (higher) expression than the current lentiviral vector in megakaryocytes and are believed to be able to restore platelet counts to normal levels in WAS patients. Additionally, the vectors described herein are believed to maintain levels of expression similar to the previous SIN LV expressing a WAS gene in all other hematopoietic cell lineages and thus is believed to be able to restore T, B and NK cell counts and function).
  • Embodiment 1 A recombinant lentiviral vector (LV) for the treatment of
  • WAS Wiskott-Aldrich Syndrome
  • an expression cassette comprising:
  • nucleic acid encoding an effective fragment of the endogenous promoter of the WAS gene where said promoter has maximum length of 600 bp and contains the sequence of HSlpro (SEQ ID NO:l);
  • WASp Wiskott-Aldrich Syndrome protein
  • Embodiment 2 The vector of embodiment 1, wherein the sequence of said effective fragment of the endogenous promoter of the WAS gene consists of the sequence of HSlpro (SEQ ID NO: 1).
  • SEQ ID NO:2 SEQ ID NOs:3-8
  • Embodiment 4 The vector of embodiment 3, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).
  • Embodiment 5 The vector of embodiment 4, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment consists of enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).
  • Embodiment 6 The vector of embodiment 3, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of the first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).
  • Embodiment 7 The vector of embodiment 6, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment consists of the first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).
  • Embodiment 8 The vector of embodiment 3, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of the 1 st half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:3).
  • Embodiment 9 The vector according to any one of embodiments 3 and 8, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of the second half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO: 4).
  • Embodiment 10 The vector according to any one of embodiments 3 and 8-9, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core Sub-Element 2 of Enhancer Element 2 (SEQ ID NO:5).
  • Embodiment 11 The vector according to any one of embodiments 3 and 8-10, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core Sub-Element 3 of Enhancer Element 2 (SEQ ID NO:6).
  • Embodiment 12 The vector according to any one of embodiments 3 and 8-11, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core-Sub Element 4 of Enhancer Element 2 (SEQ ID NO:7).
  • Embodiment 13 The vector according to any one of embodiments 3 and 8-12, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core-Sub Element 5 of Enhancer Element 2 (SEQ ID NO:8).
  • Embodiment 14 The vector according to any one of embodiments 1-13, wherein said expression cassette comprises enhancer element HS3 (SEQ ID NO:9) or an effective fragment thereof.
  • Embodiment 15 The vector of embodiment 14, wherein said expression cassette comprises an effective fragment of enhancer element HS3 wherein said fragment comprises or consists of HS3 core sequence (SEQ ID NO: 10).
  • Embodiment 16 The vector of embodiment 15, wherein said expression cassette comprises an effective fragment of enhancer element HS3 wherein said fragment consists of HS3 core sequence (SEQ ID NO: 10).
  • Embodiment 17 The vector according to any one of embodiments 1-16, wherein said expression cassette comprises enhancer element E9 (SEQ ID NO: 11) or an effective fragment thereof.
  • Embodiment 18 The vector of embodiment 17, wherein said expression cassette comprises an effective fragment of enhancer element E9 wherein said fragment comprises or consists of enhancer element E9 core sequence (SEQ ID NO: 12).
  • Embodiment 19 The vector of embodiment 18, wherein said expression cassette comprises an effective fragment of enhancer element E9 wherein said fragment consists of enhancer element E9 core sequence (SEQ ID NO: 12).
  • Embodiment 21 The vector of embodiment 20, wherein said vector comprises the features shown in Figure 18 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp)..
  • WASp Wiskott-Aldrich Syndrome protein
  • Embodiment 22 The vector of embodiment 20, wherein said vector comprises the sequence show in SEQ ID NO: 15 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
  • WASp Wiskott-Aldrich Syndrome protein
  • Embodiment 23 The vector according to any one of embodiments 1-2, wherein said expression cassette comprises: [0039] enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO: 12);
  • enhancer element HS3 sequence comprising or consisting of HS3 core sequence (SEQ ID NO: 10);
  • a fragment of the endogenous promoter of the WAS gene comprising or consisting of the sequence of HSlpro (SEQ ID NO:l).
  • Embodiment 24 The vector of embodiment 23, wherein said vector comprises the features shown in Figure 19 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
  • WASp Wiskott-Aldrich Syndrome protein
  • Embodiment 25 The vector of embodiment 23, wherein said vector comprises the sequence show in SEQ ID NO: 16 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
  • WASp Wiskott-Aldrich Syndrome protein
  • Embodiment 26 The vector according to any one of embodiments 1-2, wherein said expression cassette comprises:
  • enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO: 12);
  • enhancer element HS3 sequence comprising or consisting of HS3 core sequence (SEQ ID NO: 10);
  • enhancer element 2 core sub-element 1 SEQ ID NO:3 + SEQ ID NO:
  • enhancer element 2 core sub-element 4 SEQ ID NO:7
  • enhancer element 2 core sub-element 5 SEQ ID NO:8
  • a fragment of the endogenous promoter of the WAS gene comprising or consisting of the sequence of HSlpro (SEQ ID NO:l).
  • Embodiment 27 The vector of embodiment 26, wherein said vector comprises the features shown in Figure 20 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
  • WASp Wiskott-Aldrich Syndrome protein
  • Embodiment 28 The vector of embodiment 26, wherein said vector comprises the sequence show in SEQ ID NO: 17 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
  • WASp Wiskott-Aldrich Syndrome protein
  • Embodiment 29 The vector according to any one of embodiments 1-2, wherein said expression cassette comprises:
  • enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO: 12);
  • enhancer element HS3 sequence comprising or consisting of HS3 core sequence (SEQ ID NO: 10); a first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8); and [0055] a fragment of the endogenous promoter of the WAS gene comprising or consisting of the sequence of HSlpro (SEQ ID NO:l).
  • Embodiment 30 The vector of embodiment 29, wherein said vector comprises the features shown in Figure 21 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
  • WASp Wiskott-Aldrich Syndrome protein
  • Embodiment 31 The vector of embodiment 29, wherein said vector comprises the sequence show in SEQ ID NO: 18 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
  • WASp Wiskott-Aldrich Syndrome protein
  • Embodiment 32 The vector according to any one of embodiments 1-31, wherein said nucleic acid that encodes a nucleic acid that encodes WASp protein is a WAS cDNA or a codon-optimized WAS gene.
  • Embodiment 33 The vector of embodiment 32, wherein said nucleic acid that encodes a nucleic acid that encodes WASp protein is a WAS cDNA (SEQ ID NO: 13).
  • Embodiment 34 The vector of embodiment 32, wherein said nucleic acid that encodes a nucleic acid that encodes WASp protein is a codon optimized WAS.
  • Embodiment 35 The vector of embodiment 34, wherein the sequence of said nucleic acid that encodes WASP is a codon optimized WAS selected from the group consisting of jCAT codon optimized WAS, Gene Art optimized WAS, and IDT optimized WAS.
  • Embodiment 36 The vector according to any one of embodiments 1-35, wherein said vector comprises a y region vector genome packaging signal.
  • Embodiment 37 The vector according to any one of embodiments 1-36, wherein said vector comprise a 5' LTR comprising a CMV enhancer/promoter.
  • Embodiment 38 The vector according to any one of embodiments 1-37, wherein said vector comprises a Rev Responsive Element (RRE).
  • RRE Rev Responsive Element
  • Embodiment 39 The vector according to any one of embodiments 1-38, wherein said vector comprises a central polypurine tract.
  • Embodiment 40 The vector according to any one of embodiments 1-39, wherein said vector comprises a post-translational regulatory element.
  • Embodiment 41 The vector of embodiment 40, wherein the posttranscriptional regulatory element is modified Woodchuck Post-transcriptional Regulatory Element (WPRE).
  • WPRE Woodchuck Post-transcriptional Regulatory Element
  • Embodiment 42 The vector according to any one of embodiments 1-41, wherein said vector is incapable of reconstituting a wild-type lentivirus through recombination.
  • Embodiment 43 The vector according to any one of embodiments 1-42, wherein said vector shows high expression in megakaryocytes.
  • Embodiment 44 The vector according to any one of embodiments 1-43, wherein said vector restores T, B and NK cell counts and function when administered to a mammal having WAS.
  • Embodiment 45 A host cell transduced with a vector according to any one of embodiments 1-44.
  • Embodiment 46 The host cell of embodiment 45, wherein the cell is a stem cell.
  • Embodiment 47 The host cell of embodiment 46, wherein said cell is a stem cell derived from bone marrow, and/or from umbilical cord blood, and/or from peripheral blood.
  • Embodiment 48 The host cell of embodiment 45, wherein the cell is a human hematopoietic progenitor cell.
  • Embodiment 49 The host cell of embodiment 48, wherein the human hematopoietic progenitor cell is a CD34+ cell.
  • Embodiment 50 A method of treating Wiskott-Aldrich Syndrome (WAS), in a subject, said method comprising:
  • Embodiment 51 The method of embodiment 50, wherein the cell is a stem cell.
  • Embodiment 52 The host cell of embodiment 50, wherein said cell is a stem cell derived from bone marrow.
  • Embodiment 53 The method of embodiment 50, wherein the cell is a human hematopoietic stem and progenitor cell.
  • Embodiment 54 The method of embodiment 53, wherein the human hematopoietic progenitor cell is a CD34 + cell.
  • Embodiment 55 A recombinant nucleic acid comprising one or more of the following:
  • nucleic acid sequence comprising or consisting of a minimal endogenous promoter of the WAS gene said minimal endogenous promoter comprising or consisting of HSlpro (SEQ ID NO:l); and/or
  • nucleic acid sequence comprising or consisting of a 2nd half of Core
  • Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:4); and/or
  • nucleic acid sequence comprising or consisting of a Core Sub-
  • Element 2 of Enhancer Element 2 (SEQ ID NO: 5); and/or
  • nucleic acid sequence comprising or consisting of a Core Sub-
  • Enhancer Element 3 of Enhancer Element 2 (SEQ ID NO: 6); and/or
  • nucleic acid sequence comprising or consisting of a Core-Sub
  • Enhancer Element 4 of Enhancer Element 2 (SEQ ID NO:7);
  • nucleic acid sequence comprising or consisting of a Core-Sub
  • Enhancer Element 5 of Enhancer Element 2 (SEQ ID NO: 8); and/or
  • nucleic acid sequence comprising or consisting of enhancer element
  • E9 full (SEQ ID NO: 11) or an effective fragment thereof; and/or [0094] a nucleic acid sequence comprising or consisting of Enhancer element
  • E9 core (SEQ ID NO: 12).
  • Embodiment 56 The nucleic acid of embodiment 55, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a minimal endogenous promoter of the WAS gene said minimal endogenous promoter comprising or consisting of HSlpro (SEQ ID NO:l).
  • Embodiment 58 The nucleic acid of embodiment 55, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a 1st half of Core Sub- Element 1 of Enhancer Element 2 (SEQ ID NO: 3).
  • Embodiment 59 The nucleic acid according to any one of embodiments 55, and 58, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a 2nd half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:4).
  • Embodiment 60 The nucleic acid according to any one of embodiments 55, and 58-59, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core Sub-Element 2 of Enhancer Element 2 (SEQ ID NO:5).
  • Embodiment 61 The nucleic acid according to any one of embodiments 55, and 58-60, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core Sub-Element 3 of Enhancer Element 2 (SEQ ID NO:6).
  • Embodiment 62 The nucleic acid according to any one of embodiments 55, and 58-61, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core-Sub Element 4 of Enhancer Element 2 (SEQ ID NO:7).
  • Embodiment 63 The nucleic acid according to any one of embodiments 55, and 58-62, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core-Sub Element 5 of Enhancer Element 2 (SEQ ID NO: 8).
  • Embodiment 64 The nucleic acid according to any one of embodiments 55, and 58-63, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of enhancer element HS3 (full) (SEQ ID NO: 9) or an effective fragment thereof.
  • Embodiment 65 The nucleic acid according to any one of embodiments 55, and 58-64 , wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of Enhancer element HS3 core (SEQ ID NO: 10).
  • Embodiment 66 The nucleic acid according to any one of embodiments 55, and 58-65, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of Enhancer element E9 (full) (SEQ ID NO: 11) or an effective fragment thereof.
  • Embodiment 67 The nucleic acid according to any one of embodiments 55, and 58-66, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of Enhancer element E9 core (SEQ ID NO: 12).
  • Embodiment 68 The nucleic acid according to any one of embodiments 55-
  • nucleic acid comprises an expression cassette.
  • Embodiment 69 The nucleic acid of embodiment 68, wherein said expression cassette is effective to express WASp when transduced into a mammalian cell.
  • Embodiment 70 The nucleic acid of embodiment 55, wherein said nucleic acid comprises a vector according to any one of embodiments 1-44.
  • a “promoter” refers to a regulatory sequence in a nucleic acid required to initiate transcription of a gene (e.g., a gene operably coupled to the promoter).
  • An “enhancer” refers to a regulatory DNA sequence that, when bound by specific proteins called transcription factors, enhance the transcription of an associated gene.
  • an "effective fragment" when used with respect to a promoter refers to a fragment of the full-length promoter that is sufficient to initiate transcription of a gene operably linked to that promoter.
  • an "effective fragment" when used with respect to an enhancer refers to a fragment of the full-length enhancer that is sufficient to provide regulate expression of an operably linked gene when bound by a transcription factor. In certain embodiments the regulation is comparable with respect to expression level and/or lineage offered by the full-length enhancer.
  • operably linked refers to a nucleic acid sequence placed into a functional relationship with another nucleic acid sequence.
  • a promoter is operably linked to a gene when that promoter is placed in a location that permits that promoter to initiate transcription of that gene.
  • An enhancer is operably linked to a gene when that enhancer, when bound by an appropriate transcription factor, is able to regulate (e.g., to upregulate) expression of that gene.
  • Recombinant is used consistently with its usage in the art to refer to a nucleic acid sequence that comprises portions that do not naturally occur together as part of a single sequence or that have been rearranged relative to a naturally occurring sequence.
  • a recombinant nucleic acid is created by a process that involves the hand of man and/or is generated from a nucleic acid that was created by hand of man (e.g., by one or more cycles of replication, amplification, transcription, etc.).
  • a recombinant vims is one that comprises a recombinant nucleic acid.
  • a recombinant cell is one that comprises a recombinant nucleic acid.
  • recombinant lentiviral vector or “recombinant LV) refers to an artificially created polynucleotide vector assembled from an LV and a plurality of additional segments as a result of human intervention and manipulation.
  • an effective amount is meant the amount of a required agent or composition comprising the agent to ameliorate or eliminate symptoms of a disease relative to an untreated patient.
  • the effective amount of composition(s) used to practice the methods described herein for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
  • Figure 1 shows illustrative LV constructs used to evaluate regulatory activity of various combinations of elements HS1, HS2, HS3, and HS4.
  • Panel B shows a construct used to evaluate the combination of the E3 element and HS 1.
  • Figure 2 shows expression levels in MEG-01 cells transduced with WAS vectors.
  • Figure 3 shows expression levels of WAS vectors in Jurkat cells (T-cell line).
  • Figure 4 shows expression levels of WAS vectors in RAMOs cells (B-cell line).
  • FIG. 5 Panel A-B, illustrates the constructs used to identify the critical enhancer elements that regulate the WAS gene.
  • Panel A) Ten new constructs each containing a putative enhancer element cloned upstream of the endogenous minimal WAS promoter (HSlpro).
  • Panel B) The gamma-retroviral vector (CMMP-mCit) which was able to restore platelet counts to normal levels and used as a control.
  • CMMP-mCit The gamma-retroviral vector
  • FIG. 6 shows expression levels of WAS vectors in MEG-01
  • Figure 7 shows expression levels of WAS vectors in Jurkat cells (T-cell line).
  • Figure 8 shows expression levels of WAS vectors in RAMOs cells (B-cell line).
  • FIG. 9 shows expression levels of WAS vectors in CB CD34+ differentiated megakaryocytes "pro-megakaryocytes”.
  • Figure 10 shows expression levels of WAS vectors in CB CD34+ differentiated megakaryocytes "megakaryocytes”.
  • FIG 11 shows expression levels of WAS vectors in CB CD34+ differentiated megakaryocytes "platelets”.
  • Figure 12 WAS vectors in CB CD34+ differentiated megakaryocytes "pro megakaryocytes”.
  • Figure 13 shows expression levels of WAS vectors in CB CD34+ differentiated megakaryocytes "megakaryocytes”.
  • Figure 14 shows expression levels of WAS vectors in CB CD34+ differentiated megakaryocytes "platelets”.
  • Figure 15 shows expression levels of WAS vectors comprising each of 5 sub elements of E2 in CB CD34+ differentiated megakaryocytes "megakaryocytes”.
  • Figure 16 shows expression levels of WAS vectors comprising each of 5 E2 fragments in CB CD34+ differentiated megakaryocytes "megakaryocytes”.
  • FIG 17 shows expression levels of WAS vectors comprising each of 5 E2 fragments in in CB CD34+ differentiated megakaryocytes "platelets”.
  • Figure 18 shows a schematic of the E2(all slim) vector (E2(all slim)-HSlpro- mCit-WPRE).
  • Figure 19 shows a schematic of the E9(slim)-HS3(slim)-E2(all slim)-HSlpro- mCit-WPRE vector in which the E9(slim) and HS3(slim) elements have been added.
  • Figure 20 shows a schematic of the E9(slim)-HS3(slim)-E2(l,4,5 slim)-
  • Figure 21 shows a schematic of the E9(slim)-HS3(slim)-E2(l st half of 1 and 5 slim)-HSlpro-mCit-WPRE (Deleted 2 nd half of Core Sub-Element 1 of Element 2, Core Sub- Element 2 of Element 2 and Core Sub-Element 3 of Element 2, and Core-Sub Element 4 of Element 2).
  • Figure 22 shows expression of the candidate vectors in CB CD34+ differentiated megakaryocytes.
  • Figure 23 shows expression of the candidate vectors in CB CD34+ differentiated platelets.
  • Figure 24 shows screening of codon optimizations in an immortalized WAS patient B-cell line.
  • Figure 25 shows screening of codon optimizations in an immortalized WAS patient T-cell line.
  • Figure 26 shows titer produced by codon optimized versions of WAS Vec.
  • lentiviral vectors are provided for the treatment (or prophylaxis) of Wiskott-Aldrich Syndrome (WAS) are provided.
  • WAS Wiskott-Aldrich Syndrome
  • the vectors are optimized to reduce vector size, increase expression level and titer. Additionally, in various embodiments the vectors to recapitulate the expression pattern of the native WAS gene, e.g., as described herein.
  • a lentiviral library was constructed in order to experimentally validate each of the putative regulatory elements. Each element was validated in MEG-01 cells (Megakaryocyte cell line), Jurkats (T-cell line), RAMOs (B-cell line) as well as in a cord blood CD34+ differentiated megakaryocytes for enhancer activity. [0147] Further experiments were done to identify necessary (core) region(s) of the enhancers and the WAS endogenous promoter and the combinations of minimal promoter and reduced (slim) enhancer elements necessary to achieve suitable expression of the WAS gene were identified. The validated enhancer elements will be used to design our lead candidate lentiviral vector for the treatment of WAS.
  • a recombinant lentiviral vector (LV) for the treatment of Wiskott-Aldrich Syndrome (WAS) where the vector comprises an expression cassette comprising a nucleic acid encoding an effective fragment of the endogenous promoter of the WAS gene where the promoter has maximum length of 600 bp and contains the sequence of HSlpro (SEQ ID NO:l); and a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp) operably linked to the effective fragment of the endogenous promoter of the WAS gene.
  • the effective fragment of the endogenous promoter of the WAS gene consists of the sequence of HSlpro (SEQ ID NO:l).
  • the expression cassette comprises an effective fragment of enhancer element 2 where the fragment comprises or consists of enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).
  • the expression cassette comprises an effective fragment of enhancer element 2 where the fragment consists of enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub element 5 (SEQ ID NO: 8).
  • the expression cassette comprises an effective fragment of enhancer element 2 where the fragment comprises or consists of the first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).
  • the expression cassette comprises an effective fragment of enhancer element 2 where the fragment consists of the first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).
  • the expression cassette comprises an effective fragment of enhancer element 2 where the fragment comprises or consists of the 1 st half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:3), and/or an effective fragment of enhancer element 2 where the fragment comprises or consists of the second half of Core Sub- Element 1 of Enhancer Element 2 (SEQ ID NO:4), and/or an effective fragment of enhancer element 2 where the fragment comprises or consists of Core Sub-Element 2 of Enhancer Element 2 (SEQ ID NO:5); and/or an effective fragment of enhancer element 2 where the fragment comprises or consists of Core Sub-Element 3 of Enhancer Element 2 (SEQ ID NO:6); and/or an effective fragment of enhancer element 2 where the fragment comprises or consists of Core-Sub Element 4 of Enhancer Element 2 (SEQ ID NO:7); and/or an effective fragment of enhancer element 2 where the fragment comprises or consists of Core-Sub Element 5 of Enhancer Element 2 (SEQ ID NO: 8).
  • the expression cassette comprises enhancer element
  • the expression cassette comprises an effective fragment of enhancer element HS3 where the fragment comprises or consists of HS3 core sequence (SEQ ID NO: 10). In certain embodiments the expression cassette comprises an effective fragment of enhancer element HS3 where the fragment consists of HS3 core sequence (SEQ ID NO: 10).
  • the expression cassette comprises enhancer element
  • the expression cassette comprises an effective fragment of enhancer element E9 where the fragment comprises or consists of enhancer element E9 core sequence (SEQ ID NO: 12). In certain embodiments the expression cassette comprises an effective fragment of enhancer element E9 where the fragment consists of enhancer element E9 core sequence (SEQ ID NO: 12).
  • this vector comprises the features shown in Figure 18 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
  • this vector comprises the sequence show in SEQ ID NO: 15 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
  • the expression cassette comprises enhancer element
  • this vector comprises the features shown in Figure 19 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott- Aldrich Syndrome protein (WASp). In certain embodiments this vector comprises the sequence show in SEQ ID NO: 16 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
  • the expression cassette comprises an enhancer element
  • E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO: 12), enhancer element HS3 sequence comprising or consisting of HS3 core sequence (SEQ ID NO: 10), enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8); and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HSlpro (SEQ ID NO:l).
  • this vector comprises the features shown in Figure 20 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp). In certain embodiments this vector comprises the sequence show in SEQ ID NO: 17 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
  • the expression cassette comprises enhancer element
  • this vector comprises the features shown in Figure 21 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp). In certain embodiments this vector comprises the sequence show in SEQ ID NO: 18 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
  • the mCitrine reporter can be replaced with a nucleic acid encoding a WASp protein.
  • a nucleic acid is a WAS cDNA or a codon-optimized WAS gene (e.g., a jCAT optimized WAS).
  • the sequence of the nucleic acid that encodes WASP is a codon optimized WAS selected from the group consisting of jCAT codon optimized WAS, GeneArt optimized WAS, and IDT optimized WAS.
  • the expression cassettes described herein with respect to lentiviral vectors need not be limited to this use, and can be incorporated in essentially any other construct (e.g., a CRISPR construct) where expression of a WASp is desired.
  • nucleic acid constructs comprising any of the expression cassette components described herein are contemplated.
  • the lentiviral vectors (LVs) described herein can have various "safety" features that can include, for example, the presence of an insulator (e.g., an FB insulator in the 3'LTR). Additionally, or alternatively, in certain embodiments, the HIV LTR has been substituted with an alternative promoter (e.g., a CMV) to yield a higher titer vector without the inclusion of the HIV TAT protein during packaging. Other strong promoters (e.g., RSV, and the like can also be used).
  • an alternative promoter e.g., a CMV
  • Other strong promoters e.g., RSV, and the like can also be used.
  • the lentiviral vectors described herein contain any one or more of the elements typically found in lentiviral vectors.
  • Such elements include, but need not be limited to a y region vector genome packaging signal, a Rev Responsive Element (RRE), a polypurine tract (e.g., a central polypurine tract, a 3' polypurine tract, etc.), a post-translational regulatory element (e.g., a modified Woodchuck Post-transcriptional Regulatory Element (WPRE)), an insulator, and the like, e.g., as described below.
  • RRE Rev Responsive Element
  • PTPRE Woodchuck Post-transcriptional Regulatory Element
  • the vector is a SIN vector substantially incapable of reconstituting a wild-type lentivirus through recombination.
  • the vectors described herein shows high expression in
  • MEG-01 cells (megakaryocyte cell line), and/or in Jurkat cells (T-cell line), and/or in RAMOs cells (B-cell line).
  • the vectors described herein show high expression in CB CD34+ differentiated megakaryocytes including "pro-megakaryocytes", megakaryocytes, and platelets.
  • Example 1 As shown above, in Example 1, the vectors described herein are believed to be effective to transduce cells at high titer and to also provide high levels of expression of a nucleic acid encoding WASp protein.
  • LVs described herein e.g. , recombinant TAT-independent, SIN LVs that express a nucleic acid encoding a WASP can be used to effectively treat Wiskott-Aldrich Syndrome (WAS) in subjects (e.g., human and non-human mammals).
  • WAS Wiskott-Aldrich Syndrome
  • these vectors can be used for the modification of stem cells (e.g., hematopoietic stem and progenitor cells) that can be introduced into a subject in need thereof for the treatment of, e.g., subjects identified as having WAS.
  • the resulting cells will produce enough of the transgenic WASp protein to demonstrate significant improvement in subject health.
  • the vectors can be directly administered to a subject to achieve in vivo transduction of the target (e.g., hematopoietic stem or progenitor cells) and thereby also effect a treatment of subjects in need thereof.
  • the LVs described herein can comprise various safety features.
  • the HIV LTR has been substituted with a CMV promoter to yield higher titer vector without the inclusion of the HIV TAT protein during packaging.
  • an insulator e.g., the FB insulator
  • the LVs are also constructed to provide efficient transduction and high titer.
  • the lentiviral vector can comprise a
  • WAS gene or cDNA the nucleic acid encoding WASp is codon optimized. Numerous methods of codon optimization are known to those of skill in the art.
  • One illustrative method is JCat (Java Codon Adaptation Tool). The jCAT tool adapts gene codon usage to most sequenced prokaryotes and various eukaryotic gene expression hosts. In contrast to many tools, JCat does not require the manual definition of highly expressed genes and is, therefore, a very rapid and easy method.
  • JCat for codon adaptation
  • the output of JCat is both graphically and as Codon Adaptation Index (CAI) values given for the input sequence and the newly adapted sequence.
  • CAI Codon Adaptation Index
  • Still another codon optimization tool is IDT.
  • the IDT codon optimization tool was developed to optimize a DNA or protein sequence from one organism for expression in another by reassigning codon usage based on the frequencies of each codon’s usage in the new organism. For example, valine is encoded by 4 different codons (GUG, GUU, GUC, and GUA). In human cell lines, however, the GUG codon is preferentially used (46% use vs. 18, 24, and 12%, respectively).
  • the codon optimization tool takes this information into account and assigns valine codons with those same frequencies.
  • the tool algorithm eliminates codons with less than 10% frequency and re-normalizes the remaining frequencies to 100%.
  • the optimization tool reduces complexities that can interfere with manufacturing and downstream expression, such as repeats, hairpins, and extreme GC content.
  • the IDT optimization tool is available from IDT (Integrated DNA Technologies, Coralville, Iowa) and can be found at ww.idtdna.com/CodonOpt.
  • codon optimization tools include, but are not limited to CodonW an open source software program that can be found at codonw.sourceforge.net, and the OptimumGeneTM algorithm from GenScript.
  • the lentiviral vectors described herein comprise a TAT-independent, self-inactivating (SIN) configuration.
  • SIN TAT-independent, self-inactivating
  • SIN vectors are ones in which the production of full-length vector RNA in transduced cells is greatly reduced or abolished altogether. This feature minimizes the risk that replication- competent recombinants (RCRs) will emerge. Furthermore, it reduces the risk that that cellular coding sequences located adjacent to the vector integration site will be aberrantly expressed.
  • SIN LVs can often permit full activity of the internal promoter.
  • the SIN design increases the biosafety of the LVs. The majority of the HIV
  • the LTR is comprised of the U3 sequences.
  • the U3 region contains the enhancer and promoter elements that modulate basal and induced expression of the HIV genome in infected cells and in response to cell activation.
  • Several of these promoter elements are essential for viral replication.
  • Some of the enhancer elements are highly conserved among viral isolates and have been implicated as critical virulence factors in viral pathogenesis.
  • the enhancer elements may act to influence replication rates in the different cellular target of the vims [0175] As viral transcription starts at the 3' end of the U3 region of the 5' LTR, those sequences are not part of the viral mRNA and a copy thereof from the 3' LTR acts as template for the generation of both LTR's in the integrated provirus. If the 3' copy of the U3 region is altered in a retroviral vector construct, the vector RNA is still produced from the intact 5'
  • the retrovirus is self-inactivating (SIN) and those vectors are known as SIN transfer vectors.
  • self-inactivation is achieved through the introduction of a deletion in the U3 region of the 3' LTR of the vector DNA, /. ⁇ ? ., the DNA used to produce the vector RNA. During RT, this deletion is transferred to the 5' LTR of the proviral DNA.
  • this deletion is transferred to the 5' LTR of the proviral DNA.
  • the 5' end of the U3 region serves another essential function in vector transfer, being required for integration (terminal dinucleotide+att sequence).
  • the terminal dinucleotide and the att sequence may represent the 5' boundary of the U3 sequences which can be deleted.
  • some loosely defined regions may influence the activity of the downstream polyadenylation site in the R region. Excessive deletion of U3 sequence from the 3'LTR may decrease polyadenylation of vector transcripts with adverse consequences both on the titer of the vector in producer cells and the transgene expression in target cells.
  • the lentiviral sequences removed from the LTRs are replaced with comparable sequences from a non- lentiviral retrovirus, thereby forming hybrid LTRs.
  • the lentiviral R region within the LTR can be replaced in whole or in part by the R region from a non-lentiviral retrovirus.
  • the lentiviral TAR sequence a sequence which interacts with TAT protein to enhance viral replication, is removed, preferably in whole, from the R region.
  • the TAR sequence is then replaced with a comparable portion of the R region from a non- lentiviral retrovirus, thereby forming a hybrid R region.
  • the LTRs can be further modified to remove and/or replace with non-lentiviral sequences all or a portion of the lentiviral U3 and U5 regions.
  • the SIN configuration provides a retroviral LTR comprising a hybrid lentiviral R region that lacks all or a portion of its TAR sequence, thereby eliminating any possible activation by TAT, wherein the TAR sequence or portion thereof is replaced by a comparable portion of the R region from a non-lentiviral retrovirus, thereby forming a hybrid R region.
  • the retroviral LTR comprises a hybrid R region, wherein the hybrid R region comprises a portion of the HIV R region (e.g., a portion comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 10 in US 2003/0039636) lacking the TAR sequence, and a portion of the MoMSV R region (e.g., a portion comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 9 in 2003/0039636) comparable to the TAR sequence lacking from the HIV R region.
  • the hybrid R region comprises a portion of the HIV R region (e.g., a portion comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 10 in US 2003/0039636) lacking the TAR sequence, and a portion of the MoMSV R region (e.g., a portion comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 9 in 2003/0039636) comparable to the TAR sequence lacking from the HIV R region.
  • the entire hybrid R region comprises or consists of the nucleotide sequence shown in SEQ ID NO: 11 in 2003/0039636.
  • Suitable lentiviruses from which the R region can be derived include, for example, HIV (HIV-1 and HIV-2), EIV, SIV and FIV.
  • Suitable retroviruses from which non- lentiviral sequences can be derived include, for example, MoMSV, MoMLV, Friend, MSCV, RSV and Spumaviruses.
  • the lentivirus is HIV and the non- lentiviral retrovirus is MoMSV.
  • the LTR comprising a hybrid R region is a left (5') LTR and further comprises a promoter sequence upstream from the hybrid R region.
  • Preferred promoters are non-lentiviral in origin and include, for example, the U3 region from a non-lentiviral retrovirus (e.g., the MoMSV U3 region).
  • the U3 region comprises the nucleotide sequence shown in SEQ ID NO: 12 in US 2003/0039636.
  • the left (5') LTR further comprises a lentiviral U5 region downstream from the hybrid R region.
  • the U5 region is the HIV U5 region including the HIV att site necessary for genomic integration.
  • the U5 region comprises the nucleotide sequence shown in SEQ ID NO: 13 in US 2003/0039636.
  • the entire left (5') hybrid LTR comprises the nucleotide sequence shown in SEQ ID NO: 1 in US 2003/0039636.
  • the LTR comprising a hybrid R region is a right (3') LTR and further comprises a modified (e.g., truncated) lentiviral U3 region upstream from the hybrid R region.
  • the modified lentiviral U3 region can include the att sequence, but lack any sequences having promoter activity, thereby causing the vector to be SIN in that viral transcription cannot go beyond the first round of replication following chromosomal integration.
  • the modified lentiviral U3 region upstream from the hybrid R region consists of the 3' end of a lentiviral (e.g. , HIV) U3 region up to and including the lentiviral U3 att site.
  • the U3 region comprises the nucleotide sequence shown in SEQ ID NO: 15 in US 2003/0039636.
  • the right (3') LTR further comprises a polyadenylation sequence downstream from the hybrid R region.
  • polyadenylation sequence comprises the nucleotide sequence shown in SEQ ID NO: 16 in US 2003/0039636.
  • entire right (5') LTR comprises the nucleotide sequence shown in SEQ ID NO: 2 or 17 of US 2003/0039636.
  • the cassette expressing a nucleic acid encoding WASp is a SIN vector with the CMV enhancer/promoter substituted in the 5' LTR.
  • the CMV promoter typically provides a high level of non-tissue specific expression.
  • Other promoters with similar constitutive activity include, but are not limited to the RSV promoter, and the SV40 promoter.
  • Mammalian promoters such as the beta-actin promoter, ubiquitin C promoter, elongation factor lapromoter, tubulin promoter, etc. , may also be used.
  • LTR transcription is reduced by about 95% to about 99%.
  • LTR may be rendered at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% at least about 96%, at least about 97%, at least about 98%, or at least about 99% transcriptionally inactive.
  • insulators are inserted into the lentiviral vectors described herein.
  • Insulators are DNA sequence elements present throughout the genome. They bind proteins that modify chromatin and alter regional gene expression.
  • the placement of insulators in the vectors described herein offer various potential benefits including, inter alia : 1) Shielding of the vector from positional effect variegation of expression by flanking chromosomes (/. ⁇ ? ., barrier activity); and 2) Shielding flanking chromosomes from insertional trans- activation of gene expression by the vector (enhancer blocking).
  • insulators can help to preserve the independent function of genes or transcription units embedded in a genome or genetic context in which their expression may otherwise be influenced by regulatory signals within the genome or genetic context (see, e.g. , Burgess-Beusse el al. (2002) Proc. Natl. Acad. Sci. USA, 99: 16433; and Zhan el al. (2001) Hum. Genet., 109: 471).
  • insulators may contribute to protecting lenti virus -expressed sequences from integration site effects, which may be mediated by ex acting elements present in genomic DNA and lead to deregulated expression of transferred sequences.
  • LVs are provided in which an insulator sequence is inserted into one or both LTRs or elsewhere in the region of the vector that integrates into the cellular genome.
  • the first and best characterized vertebrate chromatin insulator is located within the chicken b-globin locus control region.
  • This element which contains a DNase-I hypersensitive site-4 (cHS4), appears to constitute the 5' boundary of the chicken b-globin locus (Prioleau el al. (1999) EMBO J. 18: 4035-4048).
  • cHS4 DNase-I hypersensitive site-4
  • a 1.2-kb fragment containing the cHS4 element displays classic insulator activities, including the ability to block the interaction of globin gene promoters and enhancers in cell lines (Chung et al. (1993) Cell, 74: 505-514), and the ability to protect expression cassettes in Drosophila (Id.), transformed cell lines (Pikaart et al.
  • FB FII/BEAD-A
  • FB FII/BEAD-A
  • FB FII/BEAD-A
  • FB 77 bp insulator element
  • the FB “synthetic” insulator has full enhancer blocking activity.
  • This insulator is illustrative and non-limiting.
  • Other suitable insulators may be used including, for example, the full-length chicken beta-globin HS4 or insulator sub-fragments thereof, the ankyrin gene insulator, and other synthetic insulator elements.
  • the vectors described herein further comprise a packaging signal.
  • a "packaging signal,” “packaging sequence,” or “PSI sequence” is any nucleic acid sequence sufficient to direct packaging of a nucleic acid whose sequence comprises the packaging signal into a retroviral particle. The term includes naturally occurring packaging sequences and also engineered variants thereof.
  • Packaging signals of a number of different retroviruses, including lentiviruses, are known in the art.
  • One illustrative, but non- limiting PSI is provided by SEQ ID NO:21.
  • the lenti viral vectors described herein comprise a Rev response element (RRE) to enhance nuclear export of unspliced RNA.
  • RREs are well known to those of skill in the art.
  • Illustrative RREs include, but are not limited to RREs such as that located at positions 7622-8459 in the HIV NL4-3 genome (Genbank accession number AF003887) as well as RREs from other strains of HIV or other retroviruses. Such sequences are readily available from Genbank or from the database with URL hiv- web.lanl.gov/content/index.
  • RRE PolvPurine Tract (cPPT, 3'PPT).
  • the lentiviral vectors described herein further include a polypurine tract (e.g., central polypurine tract (cPPT), 3' poplypurine tract (3'PPT)). Insertion of a fragment containing the 3'PPT (see, e.g., SEQ ID NO:24) or the central polypurine tract (cPPT) in lentiviral (e.g., HIV-1) vector constructs is known to enhance transduction efficiency.
  • a polypurine tract e.g., central polypurine tract (cPPT), 3' poplypurine tract (3'PPT)
  • Insertion of a fragment containing the 3'PPT (see, e.g., SEQ ID NO:24) or the central polypurine tract (cPPT) in lentiviral (e.g., HIV-1) vector constructs is known to enhance transduction efficiency.
  • the lentiviral vectors (LVs) described herein may comprise any of a variety of posttranscriptional regulatory elements (PREs) whose presence within a transcript increases expression of the heterologous nucleic acid (e.g., a nucleic acid that encodes WASp) at the protein level.
  • PREs may be particularly useful in certain embodiments, especially those that involve lentiviral constructs with modest promoters.
  • PRE One type of PRE is an intron positioned within the expression cassette, which can stimulate gene expression.
  • introns can be spliced out during the life cycle events of a lentivirus.
  • introns are typically placed in an opposite orientation to the vector genomic transcript.
  • Posttranscriptional regulatory elements that do not rely on splicing events offer the advantage of not being removed during the viral life cycle.
  • Some examples are the posttranscriptional processing element of herpes simplex vims, the posttranscriptional regulatory element of the hepatitis B vims (HPRE) and the woodchuck hepatitis virus (WPRE). Of these the WPRE is typically preferred as it contains an additional cis-acting element not found in the HPRE.
  • This regulatory element is typically positioned within the vector so as to be included in the RNA transcript of the transgene, but outside of stop codon of the transgene translational unit.
  • the WPRE is characterized and described in U.S. Pat. No: 6,136,597.
  • the WPRE is an RNA export element that mediates efficient transport of RNA from the nucleus to the cytoplasm. It enhances the expression of transgenes by insertion of a cis- acting nucleic acid sequence, such that the element and the transgene are contained within a single transcript. Presence of the WPRE in the sense orientation was shown to increase transgene expression by up to 7- to 10-fold.
  • Retroviral vectors transfer sequences in the form of cDNAs instead of complete intron-containing genes as introns are generally spliced out during the sequence of events leading to the formation of the retroviral particle.
  • Introns mediate the interaction of primary transcripts with the splicing machinery. Because the processing of RNAs by the splicing machinery facilitates their cytoplasmic export, due to a coupling between the splicing and transport machineries, cDNAs are often inefficiently expressed. Thus, the inclusion of the WPRE (see, e.g., SEQ ID NO:23) in a vector results in enhanced expression of transgenes.
  • the recombinant lentiviral vectors (LV) and resulting virus described herein are capable of transferring a heterologous nucleic acid sequence (e.g. , a nucleic acid encoding WASp) into a mammalian cell.
  • a heterologous nucleic acid sequence e.g. , a nucleic acid encoding WASp
  • vectors described herein are preferably used in conjunction with a suitable packaging cell line or co transfected into cells in vitro along with other vector plasmids containing the necessary retroviral genes (e.g., gag and pol) to form replication incompetent virions capable of packaging the vectors of the present invention and infecting cells.
  • the vectors are introduced via transfection into a packaging cell line.
  • the packaging cell line produces viral particles that contain the vector genome. Methods for transfection are well known by those of skill in the art. After cotransfection of the packaging vectors and the transfer vector to the packaging cell line, the recombinant virus is recovered from the culture media and titered by standard methods used by those of skill in the art.
  • the packaging constructs can be introduced into human cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with or without a dominant selectable marker, such as neomycin, DHFR, Glutamine synthetase, followed by selection in the presence of the appropriate drug and isolation of clones.
  • the selectable marker gene can be linked physically to the packaging genes in the construct.
  • Stable cell lines wherein the packaging functions are configured to be expressed by a suitable packaging cell are known (see, e.g., U.S. Patent No. 5,686,279, which describes packaging cells).
  • a suitable packaging cell for the production of vims particles, one may employ any cell that is compatible with the expression of lentiviral Gag and Pol genes, or any cell that can be engineered to support such expression.
  • producer cells such as 293T cells and HT1080 cells may be used.
  • the packaging cells with a lentiviral vector incorporated therein form producer cells.
  • Producer cells are thus cells or cell-lines that can produce or release packaged infectious viral particles carrying the therapeutic gene of interest (e.g., nucleic acid encoding WASp). These cells can further be anchorage dependent which means that these cells will grow, survive, or maintain function optimally when attached to a surface such as glass or plastic.
  • Some examples of anchorage dependent cell lines used as lentiviral vector packaging cell lines when the vector is replication competent are HeLa or 293 cells and PERC.6 cells.
  • methods are provided of delivering a gene to a cell which is then integrated into the genome of the cell, comprising contacting the cell with a virion containing a lentiviral vector described herein.
  • the cell e.g., in the form of tissue or an organ
  • a subject e.g. , a mammal, animal or human
  • the gene e.g., a nucleic acid encoding WASp
  • the cell can be autologous to the subject (/. ⁇ ? ., from the subject) or it can be non-autologous (/.
  • the cells can be from a wide variety including, for example, bone marrow cells, mesenchymal stem cells (e.g., obtained from adipose tissue), and other primary cells derived from human and animal sources.
  • the virion can be directly administered in vivo to a subject or a localized area of a subject (e.g., bone marrow).
  • the lentivectors described herein will be particularly useful in the transduction of human hematopoietic progenitor cells or a hematopoietic stem cells, obtained either from the bone marrow, the peripheral blood or the umbilical cord blood, as well as in the transduction of a CD4 + T cell, a peripheral blood B or T lymphocyte cell, and the like.
  • particularly preferred targets are CD34 + hematopoetic stem and progenitor cells.
  • methods for transducing a human hematopoietic stem cell.
  • the methods involve contacting a population of human cells that include hematopoietic stem cells with one of the foregoing lentivectors under conditions to effect the transduction of a human hematopoietic progenitor cell in said population by the vector.
  • the stem cells may be transduced in vivo or in vitro, depending on the ultimate application. Even in the context of human gene therapy, such as gene therapy of human stem cells, one may transduce the stem cell in vivo or, alternatively, transduce in vitro followed by infusion of the transduced stem cell into a human subject.
  • the human stem cell can be removed from a human, e.g., a WAS patient, using methods well known to those of skill in the art and transduced as noted above.
  • the transduced stem cells are then reintroduced into the same or a different human.
  • the lentivectors described herein are particularly useful for the transduction of human hematopoietic progenitor cells or haematopoietic stem cells (HSCs), obtained either from the bone marrow, the peripheral blood or the umbilical cord blood, as well as in the transduction of a CD4 + T cell, a peripheral blood B or T lymphocyte cell, and the like.
  • HSCs haematopoietic stem cells
  • the vector particles are incubated with the cells using a dose generally in the order of between 1 to 50 multiplicities of infection (MOI) which also corresponds to 1 x 10 5 to 50 x 10 5 transducing units of the viral vector per 10 5 cells.
  • MOI multiplicities of infection
  • the amount of vector may be expressed in terms of HT-29 transducing units (TU).
  • cell-based therapies involve providing stem cells and/or hematopoietic precursors, transduce the cells with the lentivirus encoding, e.g., a nucleic acid that encodes WASp, and then introduce the transformed cells into a subject in need thereof (e.g., a subject with a mutation in the WAS gene).
  • the lentivirus encoding e.g., a nucleic acid that encodes WASp
  • the methods involve isolating population of cells, e.g., stem cells from a subject, optionally expand the cells in tissue culture, and administer the lentiviral vector whose presence within a cell results in production of a normal WASp in the cells in vitro.
  • the cells are then returned to the subject, where, for example, they may provide a population of red blood cells that produce the WASp.
  • a population of cells which may be cells from a cell line or from an individual other than the subject, can be used.
  • Methods of isolating stem cells, immune system cells, etc., from a subject and returning them to the subject are well known in the art. Such methods are used, e.g., for bone marrow transplant, peripheral blood stem cell transplant, etc. , in patients undergoing chemotherapy.
  • stem cells are to be used, it will be recognized that such cells can be derived from a number of sources including bone marrow (BM), cord blood (CB), mobilized peripheral blood stem cells (mPBSC), and the like.
  • BM bone marrow
  • CB cord blood
  • mPBSC mobilized peripheral blood stem cells
  • IPCs induced pluripotent stem cells
  • HSCs hematopoietic stem cells
  • lentiviral vector described herein see, e.g., Figures
  • nucleic acid encoding WASp instead of mCit is used in stem cell gene therapy for WAS by introducing a nucleic acid that encodes WASp into the into the bone marrow stem cells of patients with WAS followed by autologous transplantation.
  • lentiviral compositions may be formulated for delivery by any available route including, but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, rectal, and vaginal. Commonly used routes of delivery include inhalation, parenteral, and transmucosal.
  • compositions can include an LV in combination with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration ⁇ Supplementary active compounds can also be incorporated into the compositions.
  • active agents i.e., a lentiviral described herein and/or other agents to be administered together the vector
  • carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, poly glycolic acid, collagen, poly orthoesters, and polylactic acid. Methods for preparation of such compositions will be apparent to those skilled in the art. Suitable materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomes can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.
  • compositions are targeted to particular cell types or to cells that are infected by a vims.
  • compositions can be targeted using monoclonal antibodies to cell surface markers, e.g., endogenous markers or viral antigens expressed on the surface of infected cells.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit comprising a predetermined quantity of a LV calculated to produce the desired therapeutic effect in association with a pharmaceutical carrier.
  • a unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.
  • Unit dose of the LV described herein may conveniently be described in terms of transducing units (T.U.) of lentivector, as defined by titering the vector on a cell line such as HeLa or 293.
  • unit doses can range from 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 s , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 T.U. and higher.
  • compositions can be administered at various intervals and over different periods of time as required, e.g., one time per week for between about 1 to about 10 weeks; between about 2 to about 8 weeks; between about 3 to about 7 weeks; about 4 weeks; about 5 weeks; about 6 weeks, etc. It may be necessary to administer the therapeutic composition on an indefinite basis.
  • the skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
  • Treatment of a subject with a LV can include a single treatment or, in many cases, can include a series of treatments.
  • LV LV
  • appropriate doses of a LV may depend upon the particular recipient and the mode of administration.
  • the appropriate dose level for any particular subject may depend upon a variety of factors including the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate: of excretion, other administered therapeutic agents, and the like.
  • lentiviral gene therapy vectors described herein can be delivered to a subject by, for example, intravenous injection, local administration, or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA, 91: 3054).
  • vectors may be delivered orally or inhalationally and may be encapsulated or otherwise manipulated to protect them from degradation, enhance uptake into tissues or cells, etc.
  • Pharmaceutical preparations can include a LV in an acceptable diluent, or can comprise a slow release matrix in which a LV is imbedded.
  • a pharmaceutical preparation can include one or more cells which produce vectors.
  • Pharmaceutical compositions comprising a LV described herein can be included in a container, pack, or dispenser, optionally together with instructions for administration.
  • compositions, methods and uses are intended to be illustrative and not limiting. Using the teachings provided herein other variations on the compositions, methods and uses will be readily available to one of skill in the art.
  • the WAS 1.6 vector is driven by a 1600bp promoter fragment immediately upstream of the transcription start site.
  • MEG-01 cells (megakaryocyte cell lines) were transduced with the WAS vectors in order to determine if any of the newly identified enhancer elements increases expression in megakaryocyte lineage. The cells were cultured for 14 days and flow cytometry was performed for expression (mCitrine) and VCN analysis.
  • the HS1 minimal promoter (417bp) appears to be the main driver of expression in the 1.6kb vector in megakaryocytes. Additionally, HS2 seems to be inert in megakaryocyte cell lines, however this element may have enhancer activity in other lineages. HS3 appears to give a major boost in expression in megakaryocyte cell lines and HS4 also appears to give a boost in expression in megakaryocyte cell lines. There is an effect of combining HS3 and HS4 and E3 is a strong non- lineage specific enhance that is also active in megakaryocytes.
  • the clinical 1.6kb vector is shown to express at curative levels in all hematopoietic cell lines except for megakaryocytes (patients are no longer immune deficient but still thrombocytopenic).
  • WAS1.6 an illustrative slim version of WAS1.6 is HS2-HS1 since HS2-HS1 contains all the regulatory regions WAS 1.6 has (while saving 1.0 kb of sequence) suggesting that this can be used as a vector backbone.
  • HS4-HS3- HS2-HS1 may provide therapeutic levels of expression in all hematopoietic cell lineages.
  • HS4-HS3-HS2-HS1 is only 155bp larger than WAS 1.6, but was shown to have ⁇ 2-fold higher expression in megakaryocytes.
  • WAS 1.6 in other cell lineages T cells and B cells. Additionally, we wished to determine if HS3 and HS4 further boost expression in other hematopoietic cell lineages, and if the HS2 enhancer element is necessary. In this regard we recognized that HS2 enhancer may have enhancer functions in other cell types or may be completely inert. Additionally, we wanted to determine if HSlpro could perform the same as WAS 1.6 and if adding HS4 and HS3 to HS2-HS1 negatively affects expression in other cell types?
  • the WAS vectors were transduced into Jurkats (T- cell line) and RAMOs (B-cell line). The cells were cultured for 14 days and expression was analyzed by flow cytometry and VCN.
  • the HS3 enhancer appears to increase expression in megakaryocyte cell lines, but is inert in B-cells and T-cells.
  • the HS4 enhancer appears to increase expression in megakaryocytes and B-cells but is inert in T-cells.
  • the E3 enhancer appears to increase expression in megakaryocytes but is inert in B and T cells (non-endogenous element).
  • one suitable WAS vector comprises HS4-HS3-
  • HS2-HSlpro This vector is only 200bp larger than WAS 1.6, but provides higher expression than WAS 1.6 in megakaryocytes, and B-cells and a similar level of expression compared to WAS 1.6 in T-cells.
  • FIG. 6 shows the data for MEG-01 (megakaryoblast cell line)
  • Figure 7 shows the data for Jurkat cells (T-cell line)
  • Figure 8 shows the results or RAMOs cells (B-cell line).
  • all 10 newly identified enhancer elements increase expression in MEG-01 cells (Megakaryoblastic cell line) at levels higher than the WAS1.6 vector and our previous lentiviral vector (HS4,3,2,1).
  • Elements 1, 3, 7, 8 and 9 each independently drive expression higher than the previous clinical g-retroviral CMMP-mCit vector.
  • All vectors express higher than the g-retroviral vector and at similar levels to WAS 1.6 in Jurkats (T-cell line), and all vectors express higher than the g -retroviral vector and at similar levels to WAS 1.6 in RAMOS (B-cell line). Additionally, element 2 increases expression over 2 fold higher compared to WAS 1.6 and CMMP-mCit.
  • FIG. 9-11 show expression levels of the WAS vectors in pro-megakaryocytes, megakaryocytes, and platelets, respectively.
  • the data from CB CD34+ differentiated megakaryocytes differ from data from MEG-01 cells (megakary oblast cell line).
  • enhancer element 2 seems to boost expression in the CB CD34+ megakaryocytes. While this construct expresses 2-fold higher than WAS 1.6, it under expresses compared to the g-retroviral construct.
  • One proposed lead vector was to comprise XXX-HS2-HSl-WASp-WPRE where XXX represents additional enhancer elements that can be added.
  • the HS2-HS1 component comprise the two functional elements within the WAS 1.6 promoter where HS1 is the main driver and HS2 provides an extra 190bp.
  • Various constructs comprising these elements and enhancer elements 1-10 were constructed and evaluated in pro-megakaryocytes (Figure 12), megakaryocytes ( Figure 13), and platelets ( Figure 14).
  • optimal vectors could include various components of one or more of enhancer regions HS3, E2, E9, and E10. It was noted that the HS3 is 531bp, E2 is 3678bp, E9 is 555 bp, and E10 is 455bp. Particularly in view of the length of enhancer region E2, it was desirable to identified smaller effective fragments of these regions. An analysis of enhancer element 2 (260kb downstream of WAS) was found to contain 5 fragments approximating about 3.7 kb.
  • E2 fragments E2-1 (first half of core sub element 1 + second half of core sub-element 1, see, e.g., Table 1, SEQ ID NOs:3 and 4), respectively), E2-2 (see, e.g., Table 1, SEQ ID NO:5) , E2-3 (see, e.g., Table 1, SEQ ID NO:6), E2-4 (see, e.g., Table 1, SEQ ID NO:7), and E2-5 (see, e.g., Table 1, SEQ ID NO:8) were cloned into 5 different vectors so that the most active fragments of E2 could be identified for inclusion in the vector.
  • vector (3) above eliminates sub-elements 2 and 3 in Enhancer element 2
  • vector (4) above eliminates the second half of sub-element 1, and sub elements 2, 3, and 4 of Enhancer element 2.
  • nucleic acid encoding the WASp protein.
  • nucleic acids include, but are not limited to a WAS cDNA, and a codon-optimized WAS nucleic acid.
  • Example 1 described the generation of four lead candidate vectors: 1)
  • one lead candidate vector is E9(slim)-HS3(slim)- l,4,5(slim) of E2-HSlpro-mCit-WPRE (WasVec) which:
  • WASVec each encoding a different codon optimized version of the WASp open reading frame as follows:
  • WASVec led to a 1.29 fold and 1.48 fold increase in expression in B cells (Figure 24) and T cells (Figure 25) respectively compared to the non-codon optimized native cDNA.
  • Figures 24, and 25 also show that GeneArt codon optimization led to a 1.13 fold and 1.20 fold increase in expression in B and T cells respectively compare to the native cDNA, while both IDT and Benchling codon optimizations led to a decrease in expression in B-cells and T- cells. None of the codon optimizations seem to significantly affect titer. Additionally, CAT and GeneArt may slightly increase titer (Figure 26).
  • one lead candidate vector is E9(slim)-

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Abstract

In certain embodiments a lentiviral vector for the treatment of Wiskott-Aldrich Syndrome (WAS) is provided. In certain embodiments the vector comprises an expression cassette comprising a nucleic acid construct comprising an effective fragment of the endogenous promoter of the WAS gene where said promoter has maximum length of 600 bp and contains the sequence of HS1pro, and a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp) operably linked to the effective fragment of the endogenous promoter of the WAS gene.

Description

LENTIVIRAL VECTORS IN HEMATOPOIETIC STEM CELLS TO TREAT WISKOTT-ALDRICH SYNDROME (WAS)
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claim priority to and benefit of USSN 62/933,875, filed on
November 11, 2019, which is incorporated herein by reference in its entirety for all purposes.
STATEMENT OF GOVERNMENTAL SUPPORT
[ Not Applicable ]
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A
TEXT FILE
[0002] A Sequence Listing is provided herewith as a text file, “UCLA-
P220P_ST25.txt” created on November 9, 2020 and having a size of 82.1 kb. The contents of the text file are incorporated by reference herein in their entirety.
BACKGROUND
[0003] Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immune deficiency caused by mutations in the Wiskott-Aldrich Syndrome (WAS) gene. Patients with WAS have severe defects in both adaptive and innate immunity and are highly susceptible to life- threatening viral and bacterial infections. Patients also suffer from extremely severe eczema, microthrombocytopenia and have a high risk of developing autoimmunity and cancer,
[0004] WAS occurs most often in males due to its X-linked recessive pattern of inheritance, affecting between 1 and 10 males per million. The first signs are usually petechiae and bruising, resulting from a low platelet count (i.e, thrombocytopenia). Spontaneous nose bleeds and bloody diarrhea are also common, and eczema typically develops within the first month of life. Recurrent bacterial infections typically develop by three months. The majority of children with WAS develop at least one autoimmune disorder, and cancers (mainly lymphoma and leukemia) develop in up to a third of patients. Immunoglobulin M (IgM) levels are typically reduced, IgA and IgE are typically elevated, and IgG levels can be normal, reduced, or elevated. In addition to thrombocytopenia, WAS patients have abnormally small platelets (i.e. microthrombocytes) and -30% also have elevated eosinophil counts (i.e., eosinophilia). [0005] Treatment for WAS typically involves prophylactic antibiotic and antiviral therapy to manage infections. Platelet transfusions and splenectomy are used to treat thrombocytopenia (low platelet counts). Additionally, blood transfusions may be required to treat anemia resulting from excessive bleeding and a protective helmet may be used to prevent brain hemorrhages which could result from head injury. Immunosuppressive treatment is utilized for autoimmune manifestations.
[0006] Because patients with WAS have abnormal T- and B-lymphocyte function, they are typically not administered live virus vaccines since there is a possibility that a vaccine strain of the virus may cause disease. Complications of chicken pox infection occur occasionally and may be prevented by early treatment following exposure with antiviral drugs, high dose immunoglobulin replacement therapy or Varicella zoster Immune Globulin (VZIG). Other “non- live” vaccinations can be given safely to patients with WAS but may not generate protective levels of antibody.
[0007] A potential curative therapy is an allogeneic hematopoietic stem cell transplantation from a HLA matched donor. However, this is not a viable option for many patients due to the unavailability of a suitable matched donor.
[0008] An alternative curative therapy is an autologous hematopoietic stem cell
(HSC) transplantation with ex vivo gene therapy. In this approach, the patient acts as their own donor, eliminating the risk of immunological complications. Successful implementation of this approach relies on the development of a lentiviral vector or CRISPR based therapy to introduce a functional copy of the gene of interest or to correct the pathogenic mutation in the patient’s HSCs.
[0009] Previous viral-based therapies utilized the CMMP-WAS g-retroviral vector.
This therapy restored function and platelet counts in all patients. However, 7/9 patients developed acute leukemia due to insertional oncogenesis (see, e.g., Braun (2014) Sci. Transl. Med. 6(227): 227ra33).
[0010] Another therapy uses a safer SIN lentiviral vector driven by a 1.6kb promoter fragment of the endogenous WAS gene. Using this vector, about 6/7 patients showed clinical improvement post gene therapy. T, B, and NK cells were functional and normal immune cell counts were restored. Additionally, there was a decrease in severity and frequency of infections and severe eczema was resolved. However, platelet counts and mean platelet size remain under normal values. The patients remained microthrombocytopenic and retained a risk of severe bleeding episodes. SUMMARY
[0011] Described herein is the development of novel lentiviral vector(s) (LVs) for the treatment of Wiskott-Aldrich Syndrome (WAS). The vectors described herein show better (higher) expression than the current lentiviral vector in megakaryocytes and are believed to be able to restore platelet counts to normal levels in WAS patients. Additionally, the vectors described herein are believed to maintain levels of expression similar to the previous SIN LV expressing a WAS gene in all other hematopoietic cell lineages and thus is believed to be able to restore T, B and NK cell counts and function).
[0012] Accordingly, various embodiments contemplated herein may include, but need not be limited to, one or more of the following:
[0013] Embodiment 1 : A recombinant lentiviral vector (LV) for the treatment of
Wiskott-Aldrich Syndrome (WAS), said vector comprising:
[0014] an expression cassette comprising:
[0015] a nucleic acid encoding an effective fragment of the endogenous promoter of the WAS gene where said promoter has maximum length of 600 bp and contains the sequence of HSlpro (SEQ ID NO:l); and
[0016] a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp) operably linked to said effective fragment of the endogenous promoter of the WAS gene.
[0017] Embodiment 2: The vector of embodiment 1, wherein the sequence of said effective fragment of the endogenous promoter of the WAS gene consists of the sequence of HSlpro (SEQ ID NO: 1).
[0018] Embodiment 3: The vector according to any one of embodiments 1-2, wherein said expression cassette comprises a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8) or an effective fragment thereof.
[0019] Embodiment 4: The vector of embodiment 3, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).
[0020] Embodiment 5: The vector of embodiment 4, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment consists of enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).
[0021] Embodiment 6: The vector of embodiment 3, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of the first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).
[0022] Embodiment 7 : The vector of embodiment 6, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment consists of the first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).
[0023] Embodiment 8: The vector of embodiment 3, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of the 1st half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:3).
[0024] Embodiment 9: The vector according to any one of embodiments 3 and 8, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of the second half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO: 4).
[0025] Embodiment 10: The vector according to any one of embodiments 3 and 8-9, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core Sub-Element 2 of Enhancer Element 2 (SEQ ID NO:5).
[0026] Embodiment 11: The vector according to any one of embodiments 3 and 8-10, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core Sub-Element 3 of Enhancer Element 2 (SEQ ID NO:6).
[0027] Embodiment 12: The vector according to any one of embodiments 3 and 8-11, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core-Sub Element 4 of Enhancer Element 2 (SEQ ID NO:7).
[0028] Embodiment 13: The vector according to any one of embodiments 3 and 8-12, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core-Sub Element 5 of Enhancer Element 2 (SEQ ID NO:8).
[0029] Embodiment 14: The vector according to any one of embodiments 1-13, wherein said expression cassette comprises enhancer element HS3 (SEQ ID NO:9) or an effective fragment thereof.
[0030] Embodiment 15: The vector of embodiment 14, wherein said expression cassette comprises an effective fragment of enhancer element HS3 wherein said fragment comprises or consists of HS3 core sequence (SEQ ID NO: 10).
[0031] Embodiment 16: The vector of embodiment 15, wherein said expression cassette comprises an effective fragment of enhancer element HS3 wherein said fragment consists of HS3 core sequence (SEQ ID NO: 10).
[0032] Embodiment 17: The vector according to any one of embodiments 1-16, wherein said expression cassette comprises enhancer element E9 (SEQ ID NO: 11) or an effective fragment thereof.
[0033] Embodiment 18: The vector of embodiment 17, wherein said expression cassette comprises an effective fragment of enhancer element E9 wherein said fragment comprises or consists of enhancer element E9 core sequence (SEQ ID NO: 12).
[0034] Embodiment 19: The vector of embodiment 18, wherein said expression cassette comprises an effective fragment of enhancer element E9 wherein said fragment consists of enhancer element E9 core sequence (SEQ ID NO: 12).
[0035] Embodiment 20: The vector according to any one of embodiments 1-2, wherein said expression cassette comprises: a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8); and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HSlpro (SEQ ID NO:l).
[0036] Embodiment 21 : The vector of embodiment 20, wherein said vector comprises the features shown in Figure 18 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp)..
[0037] Embodiment 22: The vector of embodiment 20, wherein said vector comprises the sequence show in SEQ ID NO: 15 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp)..
[0038] Embodiment 23: The vector according to any one of embodiments 1-2, wherein said expression cassette comprises: [0039] enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO: 12);
[0040] enhancer element HS3 sequence comprising or consisting of HS3 core sequence (SEQ ID NO: 10);
[0041] a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8); and
[0042] a fragment of the endogenous promoter of the WAS gene comprising or consisting of the sequence of HSlpro (SEQ ID NO:l).
[0043] Embodiment 24: The vector of embodiment 23, wherein said vector comprises the features shown in Figure 19 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
[0044] Embodiment 25: The vector of embodiment 23, wherein said vector comprises the sequence show in SEQ ID NO: 16 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
[0045] Embodiment 26: The vector according to any one of embodiments 1-2, wherein said expression cassette comprises:
[0046] enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO: 12);
[0047] enhancer element HS3 sequence comprising or consisting of HS3 core sequence (SEQ ID NO: 10);
[0048] enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID
NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8); and
[0049] a fragment of the endogenous promoter of the WAS gene comprising or consisting of the sequence of HSlpro (SEQ ID NO:l).
[0050] Embodiment 27 : The vector of embodiment 26, wherein said vector comprises the features shown in Figure 20 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
[0051] Embodiment 28: The vector of embodiment 26, wherein said vector comprises the sequence show in SEQ ID NO: 17 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
[0052] Embodiment 29: The vector according to any one of embodiments 1-2, wherein said expression cassette comprises:
[0053] enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO: 12);
[0054] enhancer element HS3 sequence comprising or consisting of HS3 core sequence (SEQ ID NO: 10); a first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8); and [0055] a fragment of the endogenous promoter of the WAS gene comprising or consisting of the sequence of HSlpro (SEQ ID NO:l).
[0056] Embodiment 30: The vector of embodiment 29, wherein said vector comprises the features shown in Figure 21 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
[0057] Embodiment 31 : The vector of embodiment 29, wherein said vector comprises the sequence show in SEQ ID NO: 18 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
[0058] Embodiment 32: The vector according to any one of embodiments 1-31, wherein said nucleic acid that encodes a nucleic acid that encodes WASp protein is a WAS cDNA or a codon-optimized WAS gene.
[0059] Embodiment 33: The vector of embodiment 32, wherein said nucleic acid that encodes a nucleic acid that encodes WASp protein is a WAS cDNA (SEQ ID NO: 13).
[0060] Embodiment 34: The vector of embodiment 32, wherein said nucleic acid that encodes a nucleic acid that encodes WASp protein is a codon optimized WAS.
[0061] Embodiment 35: The vector of embodiment 34, wherein the sequence of said nucleic acid that encodes WASP is a codon optimized WAS selected from the group consisting of jCAT codon optimized WAS, Gene Art optimized WAS, and IDT optimized WAS.
[0062] Embodiment 36: The vector according to any one of embodiments 1-35, wherein said vector comprises a y region vector genome packaging signal.
[0063] Embodiment 37: The vector according to any one of embodiments 1-36, wherein said vector comprise a 5' LTR comprising a CMV enhancer/promoter.
[0064] Embodiment 38: The vector according to any one of embodiments 1-37, wherein said vector comprises a Rev Responsive Element (RRE).
[0065] Embodiment 39: The vector according to any one of embodiments 1-38, wherein said vector comprises a central polypurine tract. [0066] Embodiment 40: The vector according to any one of embodiments 1-39, wherein said vector comprises a post-translational regulatory element.
[0067] Embodiment 41 : The vector of embodiment 40, wherein the posttranscriptional regulatory element is modified Woodchuck Post-transcriptional Regulatory Element (WPRE).
[0068] Embodiment 42: The vector according to any one of embodiments 1-41, wherein said vector is incapable of reconstituting a wild-type lentivirus through recombination.
[0069] Embodiment 43: The vector according to any one of embodiments 1-42, wherein said vector shows high expression in megakaryocytes.
[0070] Embodiment 44: The vector according to any one of embodiments 1-43, wherein said vector restores T, B and NK cell counts and function when administered to a mammal having WAS.
[0071] Embodiment 45: A host cell transduced with a vector according to any one of embodiments 1-44.
[0072] Embodiment 46: The host cell of embodiment 45, wherein the cell is a stem cell.
[0073] Embodiment 47 : The host cell of embodiment 46, wherein said cell is a stem cell derived from bone marrow, and/or from umbilical cord blood, and/or from peripheral blood.
[0074] Embodiment 48: The host cell of embodiment 45, wherein the cell is a human hematopoietic progenitor cell.
[0075] Embodiment 49: The host cell of embodiment 48, wherein the human hematopoietic progenitor cell is a CD34+ cell.
[0076] Embodiment 50: A method of treating Wiskott-Aldrich Syndrome (WAS), in a subject, said method comprising:
[0077] transducing a stem cell and/or progenitor cell from said subject with a vector according to any one of embodiments 1-44; and
[0078] transplanting said transduced cell or cells derived therefrom into said subject where said cells or derivatives therefrom express said WASp protein. [0079] Embodiment 51 : The method of embodiment 50, wherein the cell is a stem cell.
[0080] Embodiment 52: The host cell of embodiment 50, wherein said cell is a stem cell derived from bone marrow.
[0081] Embodiment 53: The method of embodiment 50, wherein the cell is a human hematopoietic stem and progenitor cell.
[0082] Embodiment 54: The method of embodiment 53, wherein the human hematopoietic progenitor cell is a CD34+ cell.
[0083] Embodiment 55: A recombinant nucleic acid comprising one or more of the following:
[0084] a nucleic acid sequence comprising or consisting of a minimal endogenous promoter of the WAS gene said minimal endogenous promoter comprising or consisting of HSlpro (SEQ ID NO:l); and/or
[0085] a nucleic acid sequence comprising or consisting of a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8) or an effective fragment thereof; a nucleic acid sequence comprising or consisting of a 1st half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO: 3) ; and/or
[0086] a nucleic acid sequence comprising or consisting of a 2nd half of Core
Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:4); and/or
[0087] a nucleic acid sequence comprising or consisting of a Core Sub-
Element 2 of Enhancer Element 2 (SEQ ID NO: 5); and/or
[0088] a nucleic acid sequence comprising or consisting of a Core Sub-
Element 3 of Enhancer Element 2 (SEQ ID NO: 6); and/or
[0089] a nucleic acid sequence comprising or consisting of a Core-Sub
Element 4 of Enhancer Element 2 (SEQ ID NO:7); and/or
[0090] a nucleic acid sequence comprising or consisting of a Core-Sub
Element 5 of Enhancer Element 2 (SEQ ID NO: 8); and/or
[0091] a nucleic acid sequence comprising or consisting of enhancer element
HS3 (full) (SEQ ID NO: 9) or an effective fragment thereof; and/or
[0092] a nucleic acid sequence comprising or consisting of Enhancer element
HS3 core (SEQ ID NO: 10); and/or
[0093] a nucleic acid sequence comprising or consisting of Enhancer element
E9 (full) (SEQ ID NO: 11) or an effective fragment thereof; and/or [0094] a nucleic acid sequence comprising or consisting of Enhancer element
E9 core (SEQ ID NO: 12).
[0095] Embodiment 56: The nucleic acid of embodiment 55, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a minimal endogenous promoter of the WAS gene said minimal endogenous promoter comprising or consisting of HSlpro (SEQ ID NO:l).
[0096] Embodiment 57: The nucleic acid of embodiment 55, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8) or an effective fragment thereof.
[0097] Embodiment 58: The nucleic acid of embodiment 55, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a 1st half of Core Sub- Element 1 of Enhancer Element 2 (SEQ ID NO: 3).
[0098] Embodiment 59: The nucleic acid according to any one of embodiments 55, and 58, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a 2nd half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:4).
[0099] Embodiment 60: The nucleic acid according to any one of embodiments 55, and 58-59, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core Sub-Element 2 of Enhancer Element 2 (SEQ ID NO:5).
[0100] Embodiment 61: The nucleic acid according to any one of embodiments 55, and 58-60, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core Sub-Element 3 of Enhancer Element 2 (SEQ ID NO:6).
[0101] Embodiment 62: The nucleic acid according to any one of embodiments 55, and 58-61, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core-Sub Element 4 of Enhancer Element 2 (SEQ ID NO:7).
[0102] Embodiment 63: The nucleic acid according to any one of embodiments 55, and 58-62, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core-Sub Element 5 of Enhancer Element 2 (SEQ ID NO: 8).
[0103] Embodiment 64: The nucleic acid according to any one of embodiments 55, and 58-63, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of enhancer element HS3 (full) (SEQ ID NO: 9) or an effective fragment thereof. [0104] Embodiment 65: The nucleic acid according to any one of embodiments 55, and 58-64 , wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of Enhancer element HS3 core (SEQ ID NO: 10).
[0105] Embodiment 66: The nucleic acid according to any one of embodiments 55, and 58-65, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of Enhancer element E9 (full) (SEQ ID NO: 11) or an effective fragment thereof.
[0106] Embodiment 67: The nucleic acid according to any one of embodiments 55, and 58-66, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of Enhancer element E9 core (SEQ ID NO: 12).
[0107] Embodiment 68: The nucleic acid according to any one of embodiments 55-
67, wherein said nucleic acid comprises an expression cassette.
[0108] Embodiment 69: The nucleic acid of embodiment 68, wherein said expression cassette is effective to express WASp when transduced into a mammalian cell.
[0109] Embodiment 70: The nucleic acid of embodiment 55, wherein said nucleic acid comprises a vector according to any one of embodiments 1-44.
Definitions.
[0110] A "promoter" refers to a regulatory sequence in a nucleic acid required to initiate transcription of a gene (e.g., a gene operably coupled to the promoter).
[0111] An "enhancer" refers to a regulatory DNA sequence that, when bound by specific proteins called transcription factors, enhance the transcription of an associated gene.
[0112] An "effective fragment" when used with respect to a promoter (e.g., an effective fragment of a WAS promoter) refers to a fragment of the full-length promoter that is sufficient to initiate transcription of a gene operably linked to that promoter.
[0113] An "effective fragment" when used with respect to an enhancer (e.g., an effective fragment of a WAS enhancer) refers to a fragment of the full-length enhancer that is sufficient to provide regulate expression of an operably linked gene when bound by a transcription factor. In certain embodiments the regulation is comparable with respect to expression level and/or lineage offered by the full-length enhancer.
[0114] The term "operably linked" refers to a nucleic acid sequence placed into a functional relationship with another nucleic acid sequence. For example, a promoter is operably linked to a gene when that promoter is placed in a location that permits that promoter to initiate transcription of that gene. An enhancer is operably linked to a gene when that enhancer, when bound by an appropriate transcription factor, is able to regulate (e.g., to upregulate) expression of that gene.
[0115] "Recombinant" is used consistently with its usage in the art to refer to a nucleic acid sequence that comprises portions that do not naturally occur together as part of a single sequence or that have been rearranged relative to a naturally occurring sequence. A recombinant nucleic acid is created by a process that involves the hand of man and/or is generated from a nucleic acid that was created by hand of man (e.g., by one or more cycles of replication, amplification, transcription, etc.). A recombinant vims is one that comprises a recombinant nucleic acid. A recombinant cell is one that comprises a recombinant nucleic acid.
[0116] As used herein, the term "recombinant lentiviral vector" or "recombinant LV) refers to an artificially created polynucleotide vector assembled from an LV and a plurality of additional segments as a result of human intervention and manipulation.
[0117] By "an effective amount" is meant the amount of a required agent or composition comprising the agent to ameliorate or eliminate symptoms of a disease relative to an untreated patient. The effective amount of composition(s) used to practice the methods described herein for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
BRIEF DESCRIPTION OF THE DRAWINGS [0118] Figure 1, panel A, shows illustrative LV constructs used to evaluate regulatory activity of various combinations of elements HS1, HS2, HS3, and HS4. Panel B shows a construct used to evaluate the combination of the E3 element and HS 1.
[0119] Figure 2 shows expression levels in MEG-01 cells transduced with WAS vectors.
[0120] Figure 3 shows expression levels of WAS vectors in Jurkat cells (T-cell line).
[0121] Figure 4 shows expression levels of WAS vectors in RAMOs cells (B-cell line).
[0122] Figure 5, panels A-B, illustrates the constructs used to identify the critical enhancer elements that regulate the WAS gene. Panel A) Ten new constructs each containing a putative enhancer element cloned upstream of the endogenous minimal WAS promoter (HSlpro). Panel B) The gamma-retroviral vector (CMMP-mCit) which was able to restore platelet counts to normal levels and used as a control.
[0123] Figure 6 shows expression levels of WAS vectors in MEG-01
(megakary oblast cell line).
[0124] Figure 7 shows expression levels of WAS vectors in Jurkat cells (T-cell line).
[0125] Figure 8 shows expression levels of WAS vectors in RAMOs cells (B-cell line).
[0126] Figure 9 shows expression levels of WAS vectors in CB CD34+ differentiated megakaryocytes "pro-megakaryocytes".
[0127] Figure 10 shows expression levels of WAS vectors in CB CD34+ differentiated megakaryocytes "megakaryocytes".
[0128] Figure 11 shows expression levels of WAS vectors in CB CD34+ differentiated megakaryocytes "platelets".
[0129] Figure 12 WAS vectors in CB CD34+ differentiated megakaryocytes "pro megakaryocytes".
[0130] Figure 13 shows expression levels of WAS vectors in CB CD34+ differentiated megakaryocytes "megakaryocytes".
[0131] Figure 14 shows expression levels of WAS vectors in CB CD34+ differentiated megakaryocytes "platelets".
[0132] Figure 15 shows expression levels of WAS vectors comprising each of 5 sub elements of E2 in CB CD34+ differentiated megakaryocytes "megakaryocytes".
[0133] Figure 16 shows expression levels of WAS vectors comprising each of 5 E2 fragments in CB CD34+ differentiated megakaryocytes "megakaryocytes".
[0134] Figure 17 shows expression levels of WAS vectors comprising each of 5 E2 fragments in in CB CD34+ differentiated megakaryocytes "platelets".
[0135] Figure 18 shows a schematic of the E2(all slim) vector (E2(all slim)-HSlpro- mCit-WPRE).
[0136] Figure 19 shows a schematic of the E9(slim)-HS3(slim)-E2(all slim)-HSlpro- mCit-WPRE vector in which the E9(slim) and HS3(slim) elements have been added. [0137] Figure 20 shows a schematic of the E9(slim)-HS3(slim)-E2(l,4,5 slim)-
HSlpro-mCit-WPRE (Deleted Core Sub-Element 2 of Element 2 and Core Sub-Element 3 of Element 2). E9slimHS3slimE2145slimHSlpro
[0138] Figure 21 shows a schematic of the E9(slim)-HS3(slim)-E2(lst half of 1 and 5 slim)-HSlpro-mCit-WPRE (Deleted 2nd half of Core Sub-Element 1 of Element 2, Core Sub- Element 2 of Element 2 and Core Sub-Element 3 of Element 2, and Core-Sub Element 4 of Element 2).
[0139] Figure 22 shows expression of the candidate vectors in CB CD34+ differentiated megakaryocytes.
[0140] Figure 23 shows expression of the candidate vectors in CB CD34+ differentiated platelets.
[0141] Figure 24 shows screening of codon optimizations in an immortalized WAS patient B-cell line.
[0142] Figure 25 shows screening of codon optimizations in an immortalized WAS patient T-cell line.
[0143] Figure 26 shows titer produced by codon optimized versions of WAS Vec.
DETAILED DESCRIPTION
[0144] In various embodiments, lentiviral vectors are provided for the treatment (or prophylaxis) of Wiskott-Aldrich Syndrome (WAS) are provided. In certain embodiments the vectors are optimized to reduce vector size, increase expression level and titer. Additionally, in various embodiments the vectors to recapitulate the expression pattern of the native WAS gene, e.g., as described herein.
[0145] In particular, a bioinformatic analysis (using publicly available databases:
Project Encode, Ensemnbl, FANTOM, VISTA Enhancer Browser, GeneHancer) was utilized to elucidate the endogenous regulatory elements of the native WAS gene. Thirteen putative endogenous enhancer elements were identified contained within a 1.1 million base pair window.
[0146] A lentiviral library was constructed in order to experimentally validate each of the putative regulatory elements. Each element was validated in MEG-01 cells (Megakaryocyte cell line), Jurkats (T-cell line), RAMOs (B-cell line) as well as in a cord blood CD34+ differentiated megakaryocytes for enhancer activity. [0147] Further experiments were done to identify necessary (core) region(s) of the enhancers and the WAS endogenous promoter and the combinations of minimal promoter and reduced (slim) enhancer elements necessary to achieve suitable expression of the WAS gene were identified. The validated enhancer elements will be used to design our lead candidate lentiviral vector for the treatment of WAS.
[0148] Accordingly, in certain embodiments a recombinant lentiviral vector (LV) for the treatment of Wiskott-Aldrich Syndrome (WAS) is provided where the vector comprises an expression cassette comprising a nucleic acid encoding an effective fragment of the endogenous promoter of the WAS gene where the promoter has maximum length of 600 bp and contains the sequence of HSlpro (SEQ ID NO:l); and a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp) operably linked to the effective fragment of the endogenous promoter of the WAS gene. In certain embodiments the effective fragment of the endogenous promoter of the WAS gene consists of the sequence of HSlpro (SEQ ID NO:l).
[0149] In certain embodiments the expression cassette comprises a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8) or an effective fragment thereof. In certain embodiments the expression cassette comprises an effective fragment of enhancer element 2 where the fragment comprises or consists of enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8). In certain embodiments the expression cassette comprises an effective fragment of enhancer element 2 where the fragment consists of enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub element 5 (SEQ ID NO: 8). In certain embodiments the expression cassette comprises an effective fragment of enhancer element 2 where the fragment comprises or consists of the first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8). In certain embodiments the expression cassette comprises an effective fragment of enhancer element 2 where the fragment consists of the first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).
[0150] In certain embodiments the expression cassette comprises an effective fragment of enhancer element 2 where the fragment comprises or consists of the 1st half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:3), and/or an effective fragment of enhancer element 2 where the fragment comprises or consists of the second half of Core Sub- Element 1 of Enhancer Element 2 (SEQ ID NO:4), and/or an effective fragment of enhancer element 2 where the fragment comprises or consists of Core Sub-Element 2 of Enhancer Element 2 (SEQ ID NO:5); and/or an effective fragment of enhancer element 2 where the fragment comprises or consists of Core Sub-Element 3 of Enhancer Element 2 (SEQ ID NO:6); and/or an effective fragment of enhancer element 2 where the fragment comprises or consists of Core-Sub Element 4 of Enhancer Element 2 (SEQ ID NO:7); and/or an effective fragment of enhancer element 2 where the fragment comprises or consists of Core-Sub Element 5 of Enhancer Element 2 (SEQ ID NO: 8).
[0151] In certain embodiments the expression cassette comprises enhancer element
HS3 (SEQ ID NO:9) or an effective fragment thereof. In certain embodiments the expression cassette comprises an effective fragment of enhancer element HS3 where the fragment comprises or consists of HS3 core sequence (SEQ ID NO: 10). In certain embodiments the expression cassette comprises an effective fragment of enhancer element HS3 where the fragment consists of HS3 core sequence (SEQ ID NO: 10).
[0152] In certain embodiments the expression cassette comprises enhancer element
E9 (SEQ ID NO: 11) or an effective fragment thereof. In certain embodiments the expression cassette comprises an effective fragment of enhancer element E9 where the fragment comprises or consists of enhancer element E9 core sequence (SEQ ID NO: 12). In certain embodiments the expression cassette comprises an effective fragment of enhancer element E9 where the fragment consists of enhancer element E9 core sequence (SEQ ID NO: 12).
[0153] In certain embodiments the expression cassette comprises a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8), and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HSlpro (SEQ ID NO:l). In certain embodiments this vector comprises the features shown in Figure 18 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp). In certain embodiments this vector comprises the sequence show in SEQ ID NO: 15 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
[0154] In certain embodiments the expression cassette comprises enhancer element
E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO: 12), enhancer element HS3 sequence comprising or consisting of HS3 core sequence (SEQ ID NO: 10), a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8); and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HSlpro (SEQ ID NO:l). In certain embodiments this vector comprises the features shown in Figure 19 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott- Aldrich Syndrome protein (WASp). In certain embodiments this vector comprises the sequence show in SEQ ID NO: 16 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
[0155] In certain embodiments the expression cassette comprises an enhancer element
E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO: 12), enhancer element HS3 sequence comprising or consisting of HS3 core sequence (SEQ ID NO: 10), enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8); and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HSlpro (SEQ ID NO:l). In certain embodiments this vector comprises the features shown in Figure 20 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp). In certain embodiments this vector comprises the sequence show in SEQ ID NO: 17 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
[0156] In certain embodiments the expression cassette comprises enhancer element
E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO: 12), enhancer element HS3 sequence comprising or consisting of HS3 core sequence (SEQ ID NO: 10), a first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8); and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HS lpro (SEQ ID NO: 1). In certain embodiments this vector comprises the features shown in Figure 21 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp). In certain embodiments this vector comprises the sequence show in SEQ ID NO: 18 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
[0157] It will be recognized that, for clinical use, the mCitrine reporter can be replaced with a nucleic acid encoding a WASp protein. In certain embodiments such a nucleic acid is a WAS cDNA or a codon-optimized WAS gene (e.g., a jCAT optimized WAS). In certain embodiments the sequence of the nucleic acid that encodes WASP is a codon optimized WAS selected from the group consisting of jCAT codon optimized WAS, GeneArt optimized WAS, and IDT optimized WAS. [0158] It will also be recognized that the expression cassettes described herein with respect to lentiviral vectors need not be limited to this use, and can be incorporated in essentially any other construct (e.g., a CRISPR construct) where expression of a WASp is desired. Thus, in certain embodiments, nucleic acid constructs comprising any of the expression cassette components described herein are contemplated.
Table 1. Nucleic acid sequences of various components of the lentiviral vectors for treatment of WAS as described herein. [0159] In various embodiments, the lentiviral vectors (LVs) described herein can have various "safety" features that can include, for example, the presence of an insulator (e.g., an FB insulator in the 3'LTR). Additionally, or alternatively, in certain embodiments, the HIV LTR has been substituted with an alternative promoter (e.g., a CMV) to yield a higher titer vector without the inclusion of the HIV TAT protein during packaging. Other strong promoters (e.g., RSV, and the like can also be used).
[0160] As noted above, in various embodiments the lentiviral vectors described herein contain any one or more of the elements typically found in lentiviral vectors. Such elements include, but need not be limited to a y region vector genome packaging signal, a Rev Responsive Element (RRE), a polypurine tract (e.g., a central polypurine tract, a 3' polypurine tract, etc.), a post-translational regulatory element (e.g., a modified Woodchuck Post-transcriptional Regulatory Element (WPRE)), an insulator, and the like, e.g., as described below.
[0161] In various embodiments the vector is a SIN vector substantially incapable of reconstituting a wild-type lentivirus through recombination.
[0162] In various embodiments the vectors described herein shows high expression in
MEG-01 cells (megakaryocyte cell line), and/or in Jurkat cells (T-cell line), and/or in RAMOs cells (B-cell line). In certain embodiments the vectors described herein show high expression in CB CD34+ differentiated megakaryocytes including "pro-megakaryocytes", megakaryocytes, and platelets.
[0163] As shown above, in Example 1, the vectors described herein are believed to be effective to transduce cells at high titer and to also provide high levels of expression of a nucleic acid encoding WASp protein.
[0164] In view of these results, it is believed that LVs described herein, e.g. , recombinant TAT-independent, SIN LVs that express a nucleic acid encoding a WASP can be used to effectively treat Wiskott-Aldrich Syndrome (WAS) in subjects (e.g., human and non-human mammals). It is believed these vectors can be used for the modification of stem cells (e.g., hematopoietic stem and progenitor cells) that can be introduced into a subject in need thereof for the treatment of, e.g., subjects identified as having WAS. Moreover, it is believed that the resulting cells will produce enough of the transgenic WASp protein to demonstrate significant improvement in subject health. It is also believed the vectors can be directly administered to a subject to achieve in vivo transduction of the target (e.g., hematopoietic stem or progenitor cells) and thereby also effect a treatment of subjects in need thereof.
[0165] As noted above, in various embodiments the LVs described herein can comprise various safety features. For example, the HIV LTR has been substituted with a CMV promoter to yield higher titer vector without the inclusion of the HIV TAT protein during packaging. In certain embodiments an insulator (e.g., the FB insulator) can be introduced into the 3'LTR for safety. The LVs are also constructed to provide efficient transduction and high titer.
[0166] It will be appreciated that the foregoing elements are illustrative and need not be limiting. In view of the teachings provided herein, suitable substitutions for these elements will be recognized by one of skill in the art and are contemplated within the scope of the teachings provided herein.
WAS codon optimization.
[0167] As noted above, in various embodiments the lentiviral vector can comprise a
WAS gene or cDNA. However, in certain embodiments the nucleic acid encoding WASp is codon optimized. Numerous methods of codon optimization are known to those of skill in the art. One illustrative method is JCat (Java Codon Adaptation Tool). The jCAT tool adapts gene codon usage to most sequenced prokaryotes and various eukaryotic gene expression hosts. In contrast to many tools, JCat does not require the manual definition of highly expressed genes and is, therefore, a very rapid and easy method. Further options of JCat for codon adaptation include the avoidance of unwanted cleavage sites for restriction enzymes and Rho-independent transcription terminators· The output of JCat is both graphically and as Codon Adaptation Index (CAI) values given for the input sequence and the newly adapted sequence. JCat optimization is described by Grote et al. (2005) Nucleic Acids Res. 33(suppl 2): W526-W531) and a JCat tool is available online at www.jcat.de.
[0168] Another codon optimization tool is provided by GeneArt (from ThermoFisher
Scientific®.
[0169] Still another codon optimization tool is IDT. The IDT codon optimization tool was developed to optimize a DNA or protein sequence from one organism for expression in another by reassigning codon usage based on the frequencies of each codon’s usage in the new organism. For example, valine is encoded by 4 different codons (GUG, GUU, GUC, and GUA). In human cell lines, however, the GUG codon is preferentially used (46% use vs. 18, 24, and 12%, respectively). The codon optimization tool takes this information into account and assigns valine codons with those same frequencies. In addition, the tool algorithm eliminates codons with less than 10% frequency and re-normalizes the remaining frequencies to 100%. Moreover, the optimization tool reduces complexities that can interfere with manufacturing and downstream expression, such as repeats, hairpins, and extreme GC content. The IDT optimization tool is available from IDT (Integrated DNA Technologies, Coralville, Iowa) and can be found at ww.idtdna.com/CodonOpt.
[0170] Other codon optimization tools include, but are not limited to CodonW an open source software program that can be found at codonw.sourceforge.net, and the OptimumGene™ algorithm from GenScript.
[0171] These codon optimizations are illustrative and non-limiting. Using the teaching provided here and in Example 1, the WAS codon usage can readily be optimized for particular applications.
TAT-Independent and Self inactivating lentiviral vectors.
[0172] To further improve safety, in various embodiments, the lentiviral vectors described herein comprise a TAT-independent, self-inactivating (SIN) configuration. Thus, in various embodiments it is desirable to employ in the LVs described herein an LTR region that has reduced promoter activity relative to wild-type LTR. Such constructs can be provided that are effectively "self-inactivating" (SIN) which provides a biosafety feature.
SIN vectors are ones in which the production of full-length vector RNA in transduced cells is greatly reduced or abolished altogether. This feature minimizes the risk that replication- competent recombinants (RCRs) will emerge. Furthermore, it reduces the risk that that cellular coding sequences located adjacent to the vector integration site will be aberrantly expressed.
[0173] Furthermore, a SIN design reduces the possibility of interference between the
LTR and the promoter that is driving the expression of the transgene. SIN LVs can often permit full activity of the internal promoter.
[0174] The SIN design increases the biosafety of the LVs. The majority of the HIV
LTR is comprised of the U3 sequences. The U3 region contains the enhancer and promoter elements that modulate basal and induced expression of the HIV genome in infected cells and in response to cell activation. Several of these promoter elements are essential for viral replication. Some of the enhancer elements are highly conserved among viral isolates and have been implicated as critical virulence factors in viral pathogenesis. The enhancer elements may act to influence replication rates in the different cellular target of the vims [0175] As viral transcription starts at the 3' end of the U3 region of the 5' LTR, those sequences are not part of the viral mRNA and a copy thereof from the 3' LTR acts as template for the generation of both LTR's in the integrated provirus. If the 3' copy of the U3 region is altered in a retroviral vector construct, the vector RNA is still produced from the intact 5'
LTR in producer cells, but cannot be regenerated in target cells. Transduction of such a vector results in the inactivation of both LTR's in the progeny virus. Thus, the retrovirus is self-inactivating (SIN) and those vectors are known as SIN transfer vectors.
[0176] In certain embodiments self-inactivation is achieved through the introduction of a deletion in the U3 region of the 3' LTR of the vector DNA, /.<?., the DNA used to produce the vector RNA. During RT, this deletion is transferred to the 5' LTR of the proviral DNA. Typically, it is desirable to eliminate enough of the U3 sequence to greatly diminish or abolish altogether the transcriptional activity of the LTR, thereby greatly diminishing or abolishing the production of full-length vector RNA in transduced cells. However, it is generally desirable to retain those elements of the LTR that are involved in polyadenylation of the viral RNA, a function typically spread out over U3, R and U5. Accordingly, in certain embodiments, it is desirable to eliminate as many of the transcriptionally important motifs from the LTR as possible while sparing the polyadenylation determinants.
[0177] The SIN design is described in detail in Zufferey et al. (1998) J Virol. 72(12):
9873-9880, and in U.S. Patent No: 5,994,136. As described therein, there are, however, limits to the extent of the deletion at the 3' LTR. First, the 5' end of the U3 region serves another essential function in vector transfer, being required for integration (terminal dinucleotide+att sequence). Thus, the terminal dinucleotide and the att sequence may represent the 5' boundary of the U3 sequences which can be deleted. In addition, some loosely defined regions may influence the activity of the downstream polyadenylation site in the R region. Excessive deletion of U3 sequence from the 3'LTR may decrease polyadenylation of vector transcripts with adverse consequences both on the titer of the vector in producer cells and the transgene expression in target cells.
[0178] Additional SIN designs are described in U.S. Patent Publication No:
2003/0039636. As described therein, in certain embodiments, the lentiviral sequences removed from the LTRs are replaced with comparable sequences from a non- lentiviral retrovirus, thereby forming hybrid LTRs. In particular, the lentiviral R region within the LTR can be replaced in whole or in part by the R region from a non-lentiviral retrovirus. In certain embodiments, the lentiviral TAR sequence, a sequence which interacts with TAT protein to enhance viral replication, is removed, preferably in whole, from the R region. The TAR sequence is then replaced with a comparable portion of the R region from a non- lentiviral retrovirus, thereby forming a hybrid R region. The LTRs can be further modified to remove and/or replace with non-lentiviral sequences all or a portion of the lentiviral U3 and U5 regions.
[0179] Accordingly, in certain embodiments, the SIN configuration provides a retroviral LTR comprising a hybrid lentiviral R region that lacks all or a portion of its TAR sequence, thereby eliminating any possible activation by TAT, wherein the TAR sequence or portion thereof is replaced by a comparable portion of the R region from a non-lentiviral retrovirus, thereby forming a hybrid R region. In a particular embodiment, the retroviral LTR comprises a hybrid R region, wherein the hybrid R region comprises a portion of the HIV R region (e.g., a portion comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 10 in US 2003/0039636) lacking the TAR sequence, and a portion of the MoMSV R region (e.g., a portion comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 9 in 2003/0039636) comparable to the TAR sequence lacking from the HIV R region.
In another particular embodiment, the entire hybrid R region comprises or consists of the nucleotide sequence shown in SEQ ID NO: 11 in 2003/0039636.
[0180] Suitable lentiviruses from which the R region can be derived include, for example, HIV (HIV-1 and HIV-2), EIV, SIV and FIV. Suitable retroviruses from which non- lentiviral sequences can be derived include, for example, MoMSV, MoMLV, Friend, MSCV, RSV and Spumaviruses. In one illustrative embodiment, the lentivirus is HIV and the non- lentiviral retrovirus is MoMSV.
[0181] In another embodiment described in US 2003/0039636, the LTR comprising a hybrid R region is a left (5') LTR and further comprises a promoter sequence upstream from the hybrid R region. Preferred promoters are non-lentiviral in origin and include, for example, the U3 region from a non-lentiviral retrovirus (e.g., the MoMSV U3 region). In one particular embodiment, the U3 region comprises the nucleotide sequence shown in SEQ ID NO: 12 in US 2003/0039636. In another embodiment, the left (5') LTR further comprises a lentiviral U5 region downstream from the hybrid R region. In one embodiment, the U5 region is the HIV U5 region including the HIV att site necessary for genomic integration. In another embodiment, the U5 region comprises the nucleotide sequence shown in SEQ ID NO: 13 in US 2003/0039636. In yet another embodiment, the entire left (5') hybrid LTR comprises the nucleotide sequence shown in SEQ ID NO: 1 in US 2003/0039636. [0182] In another illustrative embodiment, the LTR comprising a hybrid R region is a right (3') LTR and further comprises a modified (e.g., truncated) lentiviral U3 region upstream from the hybrid R region. The modified lentiviral U3 region can include the att sequence, but lack any sequences having promoter activity, thereby causing the vector to be SIN in that viral transcription cannot go beyond the first round of replication following chromosomal integration. In a particular embodiment, the modified lentiviral U3 region upstream from the hybrid R region consists of the 3' end of a lentiviral (e.g. , HIV) U3 region up to and including the lentiviral U3 att site. In one embodiment, the U3 region comprises the nucleotide sequence shown in SEQ ID NO: 15 in US 2003/0039636. In another embodiment, the right (3') LTR further comprises a polyadenylation sequence downstream from the hybrid R region. In another embodiment, the polyadenylation sequence comprises the nucleotide sequence shown in SEQ ID NO: 16 in US 2003/0039636. In yet another embodiment, the entire right (5') LTR comprises the nucleotide sequence shown in SEQ ID NO: 2 or 17 of US 2003/0039636.
[0183] Thus, in the case of HIV based LV, it has been discovered that such vectors tolerate significant U3 deletions, including the removal of the LTR TATA box (e.g. , deletions from -418 to -18), without significant reductions in vector titers. These deletions render the LTR region substantially transcriptionally inactive in that the transcriptional ability of the LTR in reduced to about 90% or lower.
[0184] It has also been demonstrated that the trans- acting function of Tat becomes dispensable if part of the upstream LTR in the transfer vector construct is replaced by constitutively active promoter sequences (see, e.g., Dull et al. (1998) J Virol. 72(11): 8463- 8471. Furthermore, we show that the expression of rev in trans allows the production of high-titer HIV-derived vector stocks from a packaging construct which contains only gag and pol. This design makes the expression of the packaging functions conditional on complementation available only in producer cells. The resulting gene delivery system, conserves only three of the nine genes of HIV- 1 and relies on four separate transcriptional units for the production of transducing particles.
[0185] In one embodiments illustrated in Example 1, the cassette expressing a nucleic acid encoding WASp is a SIN vector with the CMV enhancer/promoter substituted in the 5' LTR.
[0186] It will be recognized that the CMV promoter typically provides a high level of non-tissue specific expression. Other promoters with similar constitutive activity include, but are not limited to the RSV promoter, and the SV40 promoter. Mammalian promoters such as the beta-actin promoter, ubiquitin C promoter, elongation factor lapromoter, tubulin promoter, etc. , may also be used.
[0187] The foregoing SIN configurations are illustrative and non-limiting. Numerous
SIN configurations are known to those of skill in the art. As indicated above, in certain embodiments, the LTR transcription is reduced by about 95% to about 99%. In certain embodiments LTR may be rendered at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% at least about 96%, at least about 97%, at least about 98%, or at least about 99% transcriptionally inactive.
Insulator element
[0188] In certain embodiments, to further enhance biosafety, insulators are inserted into the lentiviral vectors described herein. Insulators are DNA sequence elements present throughout the genome. They bind proteins that modify chromatin and alter regional gene expression. The placement of insulators in the vectors described herein offer various potential benefits including, inter alia : 1) Shielding of the vector from positional effect variegation of expression by flanking chromosomes (/.<?., barrier activity); and 2) Shielding flanking chromosomes from insertional trans- activation of gene expression by the vector (enhancer blocking). Thus, insulators can help to preserve the independent function of genes or transcription units embedded in a genome or genetic context in which their expression may otherwise be influenced by regulatory signals within the genome or genetic context (see, e.g. , Burgess-Beusse el al. (2002) Proc. Natl. Acad. Sci. USA, 99: 16433; and Zhan el al. (2001) Hum. Genet., 109: 471). In the present context insulators may contribute to protecting lenti virus -expressed sequences from integration site effects, which may be mediated by ex acting elements present in genomic DNA and lead to deregulated expression of transferred sequences. In various embodiments LVs are provided in which an insulator sequence is inserted into one or both LTRs or elsewhere in the region of the vector that integrates into the cellular genome.
[0189] The first and best characterized vertebrate chromatin insulator is located within the chicken b-globin locus control region. This element, which contains a DNase-I hypersensitive site-4 (cHS4), appears to constitute the 5' boundary of the chicken b-globin locus (Prioleau el al. (1999) EMBO J. 18: 4035-4048). A 1.2-kb fragment containing the cHS4 element displays classic insulator activities, including the ability to block the interaction of globin gene promoters and enhancers in cell lines (Chung et al. (1993) Cell, 74: 505-514), and the ability to protect expression cassettes in Drosophila (Id.), transformed cell lines (Pikaart et al. (1998) Genes Dev. 12: 2852-2862), and transgenic am als (Wang et al. (1997) Nat. Biotechnol., 15: 239-243; Taboit-Dameron et al. (1999) Transgenic Res., 8: 223- 235) from position effects. Much of this activity is contained in a 250-bp fragment. Within this stretch is a 49-bp cHS4 core (Chung et al. (1997) Proc. Natl. Acad. Sci., USA, 94: 575- 580) that interacts with the zinc finger DNA binding protein CTCF implicated in enhancer blocking assays (Bell et al. (1999) Cell, 98: 387-396).
[0190] One illustrative and suitable insulator is FB (FII/BEAD-A), a 77 bp insulator element, that contains the minimal CTCF binding site enhancer-blocking components of the chicken b-globin 5' HS4 insulators and a homologous region from the human T-cell receptor alpha/delta blocking element alpha/delta I (BEAD-I) insulator described by Ramezani et al. (2008) Stem Cell 26: 3257-3266. The FB “synthetic” insulator has full enhancer blocking activity. This insulator is illustrative and non-limiting. Other suitable insulators may be used including, for example, the full-length chicken beta-globin HS4 or insulator sub-fragments thereof, the ankyrin gene insulator, and other synthetic insulator elements.
Packaging signal.
[0191] In various embodiments the vectors described herein further comprise a packaging signal. A "packaging signal," "packaging sequence," or "PSI sequence" is any nucleic acid sequence sufficient to direct packaging of a nucleic acid whose sequence comprises the packaging signal into a retroviral particle. The term includes naturally occurring packaging sequences and also engineered variants thereof. Packaging signals of a number of different retroviruses, including lentiviruses, are known in the art. One illustrative, but non- limiting PSI is provided by SEQ ID NO:21.
Rev Responsive Element (RRE).
[0192] In certain embodiments the lenti viral vectors described herein comprise a Rev response element (RRE) to enhance nuclear export of unspliced RNA. RREs are well known to those of skill in the art. Illustrative RREs include, but are not limited to RREs such as that located at positions 7622-8459 in the HIV NL4-3 genome (Genbank accession number AF003887) as well as RREs from other strains of HIV or other retroviruses. Such sequences are readily available from Genbank or from the database with URL hiv- web.lanl.gov/content/index. One illustrative, but non-limiting RRE is shown in SEQ ID NO:22). PolvPurine Tract (cPPT, 3'PPT).
[0193] In various embodiments the lentiviral vectors described herein further include a polypurine tract (e.g., central polypurine tract (cPPT), 3' poplypurine tract (3'PPT)). Insertion of a fragment containing the 3'PPT (see, e.g., SEQ ID NO:24) or the central polypurine tract (cPPT) in lentiviral (e.g., HIV-1) vector constructs is known to enhance transduction efficiency.
Expression-Stimulating Posttranscriptional Regulatory Element (PRE)
[0194] In certain embodiments the lentiviral vectors (LVs) described herein may comprise any of a variety of posttranscriptional regulatory elements (PREs) whose presence within a transcript increases expression of the heterologous nucleic acid (e.g., a nucleic acid that encodes WASp) at the protein level. PREs may be particularly useful in certain embodiments, especially those that involve lentiviral constructs with modest promoters.
[0195] One type of PRE is an intron positioned within the expression cassette, which can stimulate gene expression. However, introns can be spliced out during the life cycle events of a lentivirus. Hence, if introns are used as PRE's they are typically placed in an opposite orientation to the vector genomic transcript.
[0196] Posttranscriptional regulatory elements that do not rely on splicing events offer the advantage of not being removed during the viral life cycle. Some examples are the posttranscriptional processing element of herpes simplex vims, the posttranscriptional regulatory element of the hepatitis B vims (HPRE) and the woodchuck hepatitis virus (WPRE). Of these the WPRE is typically preferred as it contains an additional cis-acting element not found in the HPRE. This regulatory element is typically positioned within the vector so as to be included in the RNA transcript of the transgene, but outside of stop codon of the transgene translational unit.
[0197] The WPRE is characterized and described in U.S. Pat. No: 6,136,597. As described therein, the WPRE is an RNA export element that mediates efficient transport of RNA from the nucleus to the cytoplasm. It enhances the expression of transgenes by insertion of a cis- acting nucleic acid sequence, such that the element and the transgene are contained within a single transcript. Presence of the WPRE in the sense orientation was shown to increase transgene expression by up to 7- to 10-fold. Retroviral vectors transfer sequences in the form of cDNAs instead of complete intron-containing genes as introns are generally spliced out during the sequence of events leading to the formation of the retroviral particle. Introns mediate the interaction of primary transcripts with the splicing machinery. Because the processing of RNAs by the splicing machinery facilitates their cytoplasmic export, due to a coupling between the splicing and transport machineries, cDNAs are often inefficiently expressed. Thus, the inclusion of the WPRE (see, e.g., SEQ ID NO:23) in a vector results in enhanced expression of transgenes.
Transduced Host Cells and Methods of cell transduction.
[0198] The recombinant lentiviral vectors (LV) and resulting virus described herein are capable of transferring a heterologous nucleic acid sequence (e.g. , a nucleic acid encoding WASp) into a mammalian cell. In various embodiments, for delivery to cells, vectors described herein are preferably used in conjunction with a suitable packaging cell line or co transfected into cells in vitro along with other vector plasmids containing the necessary retroviral genes (e.g., gag and pol) to form replication incompetent virions capable of packaging the vectors of the present invention and infecting cells.
[0199] In certain embodiments the vectors are introduced via transfection into a packaging cell line. The packaging cell line produces viral particles that contain the vector genome. Methods for transfection are well known by those of skill in the art. After cotransfection of the packaging vectors and the transfer vector to the packaging cell line, the recombinant virus is recovered from the culture media and titered by standard methods used by those of skill in the art. Thus, the packaging constructs can be introduced into human cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with or without a dominant selectable marker, such as neomycin, DHFR, Glutamine synthetase, followed by selection in the presence of the appropriate drug and isolation of clones. In certain embodiments the selectable marker gene can be linked physically to the packaging genes in the construct.
[0200] Stable cell lines wherein the packaging functions are configured to be expressed by a suitable packaging cell are known (see, e.g., U.S. Patent No. 5,686,279, which describes packaging cells). In general, for the production of vims particles, one may employ any cell that is compatible with the expression of lentiviral Gag and Pol genes, or any cell that can be engineered to support such expression. For example, producer cells such as 293T cells and HT1080 cells may be used.
[0201] The packaging cells with a lentiviral vector incorporated therein form producer cells. Producer cells are thus cells or cell-lines that can produce or release packaged infectious viral particles carrying the therapeutic gene of interest (e.g., nucleic acid encoding WASp). These cells can further be anchorage dependent which means that these cells will grow, survive, or maintain function optimally when attached to a surface such as glass or plastic. Some examples of anchorage dependent cell lines used as lentiviral vector packaging cell lines when the vector is replication competent are HeLa or 293 cells and PERC.6 cells.
[0202] Accordingly, in certain embodiments, methods are provided of delivering a gene to a cell which is then integrated into the genome of the cell, comprising contacting the cell with a virion containing a lentiviral vector described herein. The cell (e.g., in the form of tissue or an organ) can be contacted (e.g. , infected) with the virion ex vivo and then delivered to a subject (e.g. , a mammal, animal or human) in which the gene (e.g., a nucleic acid encoding WASp) will be expressed. In various embodiments the cell can be autologous to the subject (/.<?., from the subject) or it can be non-autologous (/.<?., allogeneic or xenogenic) to the subject. Moreover, because the vectors described herein are capable of being delivered to both dividing and non-dividing cells, the cells can be from a wide variety including, for example, bone marrow cells, mesenchymal stem cells (e.g., obtained from adipose tissue), and other primary cells derived from human and animal sources. Alternatively, the virion can be directly administered in vivo to a subject or a localized area of a subject (e.g., bone marrow).
[0203] In certain embodiments, the lentivectors described herein will be particularly useful in the transduction of human hematopoietic progenitor cells or a hematopoietic stem cells, obtained either from the bone marrow, the peripheral blood or the umbilical cord blood, as well as in the transduction of a CD4+ T cell, a peripheral blood B or T lymphocyte cell, and the like. In certain embodiments particularly preferred targets are CD34+ hematopoetic stem and progenitor cells.
Gene therapy.
[0204] In still other embodiments, methods are provided for transducing a human hematopoietic stem cell. In certain embodiments the methods involve contacting a population of human cells that include hematopoietic stem cells with one of the foregoing lentivectors under conditions to effect the transduction of a human hematopoietic progenitor cell in said population by the vector. The stem cells may be transduced in vivo or in vitro, depending on the ultimate application. Even in the context of human gene therapy, such as gene therapy of human stem cells, one may transduce the stem cell in vivo or, alternatively, transduce in vitro followed by infusion of the transduced stem cell into a human subject. In one aspect of this embodiment, the human stem cell can be removed from a human, e.g., a WAS patient, using methods well known to those of skill in the art and transduced as noted above. The transduced stem cells are then reintroduced into the same or a different human.
Stem cell/progenitor cell gene therapy.
[0205] In various embodiments the lentivectors described herein are particularly useful for the transduction of human hematopoietic progenitor cells or haematopoietic stem cells (HSCs), obtained either from the bone marrow, the peripheral blood or the umbilical cord blood, as well as in the transduction of a CD4+ T cell, a peripheral blood B or T lymphocyte cell, and the like. In certain embodiments particularly preferred targets are CD34+ hematopoietic stem and progenitor cells.
[0206] When cells, for instance CD34+ cells, dendritic cells, peripheral blood cells or tumor cells are transduced ex vivo, the vector particles are incubated with the cells using a dose generally in the order of between 1 to 50 multiplicities of infection (MOI) which also corresponds to 1 x 105 to 50 x 105 transducing units of the viral vector per 105 cells. This can include amounts of vector corresponding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, and 50 MOI. Typically, the amount of vector may be expressed in terms of HT-29 transducing units (TU).
[0207] In certain embodiments cell-based therapies involve providing stem cells and/or hematopoietic precursors, transduce the cells with the lentivirus encoding, e.g., a nucleic acid that encodes WASp, and then introduce the transformed cells into a subject in need thereof (e.g., a subject with a mutation in the WAS gene).
[0208] In certain embodiments the methods involve isolating population of cells, e.g., stem cells from a subject, optionally expand the cells in tissue culture, and administer the lentiviral vector whose presence within a cell results in production of a normal WASp in the cells in vitro. The cells are then returned to the subject, where, for example, they may provide a population of red blood cells that produce the WASp.
[0209] In some illustrative, but non-limiting, embodiments, a population of cells, which may be cells from a cell line or from an individual other than the subject, can be used. Methods of isolating stem cells, immune system cells, etc., from a subject and returning them to the subject are well known in the art. Such methods are used, e.g., for bone marrow transplant, peripheral blood stem cell transplant, etc. , in patients undergoing chemotherapy.
[0210] Where stem cells are to be used, it will be recognized that such cells can be derived from a number of sources including bone marrow (BM), cord blood (CB), mobilized peripheral blood stem cells (mPBSC), and the like. In certain embodiments the use of induced pluripotent stem cells (IPSCs) is contemplated. Methods of isolating hematopoietic stem cells (HSCs), transducing such cells and introducing them into a mammalian subject are well known to those of skill in the art.
[0211] In certain embodiments a lentiviral vector described herein (see, e.g., Figures
18-21 with a nucleic acid encoding WASp instead of mCit is used in stem cell gene therapy for WAS by introducing a nucleic acid that encodes WASp into the into the bone marrow stem cells of patients with WAS followed by autologous transplantation.
Direct introduction of vector.
[0212] In certain embodiments direct treatment of a subject by direct introduction of the vector(s) described herein is contemplated. The lentiviral compositions may be formulated for delivery by any available route including, but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, rectal, and vaginal. Commonly used routes of delivery include inhalation, parenteral, and transmucosal.
[0213] In various embodiments pharmaceutical compositions can include an LV in combination with a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration· Supplementary active compounds can also be incorporated into the compositions.
[0214] In some embodiments, active agents, i.e., a lentiviral described herein and/or other agents to be administered together the vector, are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, poly glycolic acid, collagen, poly orthoesters, and polylactic acid. Methods for preparation of such compositions will be apparent to those skilled in the art. Suitable materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomes can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.
4,522,811. In some embodiments the composition is targeted to particular cell types or to cells that are infected by a vims. For example, compositions can be targeted using monoclonal antibodies to cell surface markers, e.g., endogenous markers or viral antigens expressed on the surface of infected cells.
[0215] It is advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit comprising a predetermined quantity of a LV calculated to produce the desired therapeutic effect in association with a pharmaceutical carrier.
[0216] A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. Unit dose of the LV described herein may conveniently be described in terms of transducing units (T.U.) of lentivector, as defined by titering the vector on a cell line such as HeLa or 293. In certain embodiments unit doses can range from 103, 104, 105, 106, 107, 10s, 109, 1010, 1011, 1012, 1013 T.U. and higher.
[0217] Pharmaceutical compositions can be administered at various intervals and over different periods of time as required, e.g., one time per week for between about 1 to about 10 weeks; between about 2 to about 8 weeks; between about 3 to about 7 weeks; about 4 weeks; about 5 weeks; about 6 weeks, etc. It may be necessary to administer the therapeutic composition on an indefinite basis. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Treatment of a subject with a LV can include a single treatment or, in many cases, can include a series of treatments.
[0218] Illustrative, but non-limiting, doses for administration of gene therapy vectors and methods for determining suitable doses are known in the art. It is furthermore understood that appropriate doses of a LV may depend upon the particular recipient and the mode of administration. The appropriate dose level for any particular subject may depend upon a variety of factors including the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate: of excretion, other administered therapeutic agents, and the like.
[0219] In certain embodiments lentiviral gene therapy vectors described herein can be delivered to a subject by, for example, intravenous injection, local administration, or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA, 91: 3054). In certain embodiments vectors may be delivered orally or inhalationally and may be encapsulated or otherwise manipulated to protect them from degradation, enhance uptake into tissues or cells, etc. Pharmaceutical preparations can include a LV in an acceptable diluent, or can comprise a slow release matrix in which a LV is imbedded. Alternatively or additionally, where a vector can be produced intact from recombinant cells, as is the case for retroviral or lentiviral vectors as described herein, a pharmaceutical preparation can include one or more cells which produce vectors. Pharmaceutical compositions comprising a LV described herein can be included in a container, pack, or dispenser, optionally together with instructions for administration.
[0220] The foregoing compositions, methods and uses are intended to be illustrative and not limiting. Using the teachings provided herein other variations on the compositions, methods and uses will be readily available to one of skill in the art.
Example 1
Development of Lentiviral Vectors for Treatment of Wiskott-Aldrich Syndrome (WAS) [0221] The goal of the experiments described below was to develop a novel lentiviral vector driven by endogenous regulatory elements of the native WAS gene for the treatment of Wiskott-Aldrich Syndrome. In particular, it was desired to develop a vector that has higher expression than the current WAS 1.6 lentiviral vector in megakaryocytes and consequently able to restore platelet counts to normal levels in WAS patients. Additionally, it was desired to maintain at least a similar level of expression in all other hematopoietic cell lineages and to restore T, B and NK cell counts and function.
[0222] A bioinformatic analysis (using publicly available databases: Project Encode,
Ensemnbl, FANTOM, VISTA Enhancer Browser, GeneHancer) was utilized to elucidate the endogenous regulatory elements of the native WAS gene.
[0223] The WAS 1.6 vector is driven by a 1600bp promoter fragment immediately upstream of the transcription start site. We identified two regulatory elements "HS 1 " and "HS2" within this fragment which are 417bp and 190bp, respectively, in size. Thus, we identified 1026bp of inert sequence in the 1633bp fragment which could be removed to decrease promoter size.
[0224] We note that the 1.6kb promoter is insufficient to drive wildtype levels of expression in megakaryocytes and as a result patients remain microthrombocytopenic. In view of this we hypothesized that additional endogenous enhancer elements are necessary to drive wildtype levels of expression of WAS in megakaryocytes. [0225] A proximal analysis of the WAS gene identified new enhancer elements "HS4" and "HS3” that contain megakaryocyte DNasel HS sites and that may act as enhancers to boost expression in megakaryocytes. A series of lenti viral vectors (LVs) containing various combinations of these regions and a reporter gene were created to evaluate activity of these regulatory elements (see, e.g., Figure 1, panel A). Additionally the E3 element (Thurman et al. (2012) Nature, 489: 75-82), proposed to be one of the strongest enhancers in the human genome has megakaryocyte hypersensitivity. Accordingly, we also constructed a vector to evaluate E3 enhancer activity in this context (see, e.g., Figure 1, panel B).
[0226] MEG-01 cells (megakaryocyte cell lines) were transduced with the WAS vectors in order to determine if any of the newly identified enhancer elements increases expression in megakaryocyte lineage. The cells were cultured for 14 days and flow cytometry was performed for expression (mCitrine) and VCN analysis.
[0227] As shown in Figure 2 the HS1 minimal promoter (417bp) appears to be the main driver of expression in the 1.6kb vector in megakaryocytes. Additionally, HS2 seems to be inert in megakaryocyte cell lines, however this element may have enhancer activity in other lineages. HS3 appears to give a major boost in expression in megakaryocyte cell lines and HS4 also appears to give a boost in expression in megakaryocyte cell lines. There is an effect of combining HS3 and HS4 and E3 is a strong non- lineage specific enhance that is also active in megakaryocytes.
[0228] The clinical 1.6kb vector is shown to express at curative levels in all hematopoietic cell lines except for megakaryocytes (patients are no longer immune deficient but still thrombocytopenic). As we show, an illustrative slim version of WAS1.6 is HS2-HS1 since HS2-HS1 contains all the regulatory regions WAS 1.6 has (while saving 1.0 kb of sequence) suggesting that this can be used as a vector backbone. Adding the novel HS3 and HS4 fragment has been shown to boost expression in megakaryocytes therefore HS4-HS3- HS2-HS1 may provide therapeutic levels of expression in all hematopoietic cell lineages.
One illustrative suitable vector (HS4-HS3-HS2-HS1) is only 155bp larger than WAS 1.6, but was shown to have ~2-fold higher expression in megakaryocytes.
[0229] However, it remained to be determined if HS2-HSlpro performs the same as
WAS 1.6 in other cell lineages (T cells and B cells). Additionally, we wished to determine if HS3 and HS4 further boost expression in other hematopoietic cell lineages, and if the HS2 enhancer element is necessary. In this regard we recognized that HS2 enhancer may have enhancer functions in other cell types or may be completely inert. Additionally, we wanted to determine if HSlpro could perform the same as WAS 1.6 and if adding HS4 and HS3 to HS2-HS1 negatively affects expression in other cell types?
[0230] To address these questions, the WAS vectors were transduced into Jurkats (T- cell line) and RAMOs (B-cell line). The cells were cultured for 14 days and expression was analyzed by flow cytometry and VCN.
[0231] Expression levels of the WAS vectors in Jurkat cells are shown in Figure 3 and the expression levels in RAMOs cells are show in Figure 4. As illustrated by these figures, the bare minimal promoter HSlpro (SEQ NO:l) functions the same as WAS1.6 in all cell lines test (MEG-01, CMK, Jurkats and RAMOs). The HS2 enhancer, by itself, seems to be relatively inert (190bp). By eliminating the inert 1 kb sequence and the 190bp HS2 sequence we can save up to about 1.2kb of sequence.
[0232] The HS3 enhancer appears to increase expression in megakaryocyte cell lines, but is inert in B-cells and T-cells. The HS4 enhancer appears to increase expression in megakaryocytes and B-cells but is inert in T-cells. The E3 enhancer appears to increase expression in megakaryocytes but is inert in B and T cells (non-endogenous element).
[0233] In view of these discoveries, one suitable WAS vector comprises HS4-HS3-
HS2-HSlpro. This vector is only 200bp larger than WAS 1.6, but provides higher expression than WAS 1.6 in megakaryocytes, and B-cells and a similar level of expression compared to WAS 1.6 in T-cells.
[0234] We proceed to reanalyze the WAS locus in depth and identified 13 putative endogenous enhancer elements contained within a 1.1 million base pair window spanning 850kb upstream and 250kb downstream of the WAS gene. Three of the elements were previously identified in our proximal bioinformatic analysis of the WAS locus. In order to experimentally identify the critical enhancer elements that regulate the WAS gene, each putative enhancer element was cloned upstream of the endogenous minimal WAS promoter (HSlpro) to drive expression of mCitrine (see, Figure 5, panel A). The expression of these vectors was compared to the gamma-retroviral vector (CMMP-mCit, see, Figure 5, panel B) used in previous controls and which was able to restore platelet counts to normal levels.
[0235] Cells were transduced with the vectors, cultured for 14 days and expression was analyzed by flow cytometry and VCN. Figure 6 shows the data for MEG-01 (megakaryoblast cell line), Figure 7 shows the data for Jurkat cells (T-cell line), and Figure 8 shows the results or RAMOs cells (B-cell line). As shown in these Figures, all 10 newly identified enhancer elements increase expression in MEG-01 cells (Megakaryoblastic cell line) at levels higher than the WAS1.6 vector and our previous lentiviral vector (HS4,3,2,1). Elements 1, 3, 7, 8 and 9 each independently drive expression higher than the previous clinical g-retroviral CMMP-mCit vector. All vectors express higher than the g-retroviral vector and at similar levels to WAS 1.6 in Jurkats (T-cell line), and all vectors express higher than the g -retroviral vector and at similar levels to WAS 1.6 in RAMOS (B-cell line). Additionally, element 2 increases expression over 2 fold higher compared to WAS 1.6 and CMMP-mCit.
[0236] The 10 elements were screened again in cord blood (CB) CD34+ HSPCs differentiated into megakaryocytes and platelets to confirm result from the cell lines. Figures 9-11 show expression levels of the WAS vectors in pro-megakaryocytes, megakaryocytes, and platelets, respectively. As shown in these figures, the data from CB CD34+ differentiated megakaryocytes differ from data from MEG-01 cells (megakary oblast cell line). In particular, only enhancer element 2 seems to boost expression in the CB CD34+ megakaryocytes. While this construct expresses 2-fold higher than WAS 1.6, it under expresses compared to the g-retroviral construct.
[0237] One proposed lead vector was to comprise XXX-HS2-HSl-WASp-WPRE where XXX represents additional enhancer elements that can be added. The HS2-HS1 component comprise the two functional elements within the WAS 1.6 promoter where HS1 is the main driver and HS2 provides an extra 190bp. Various constructs comprising these elements and enhancer elements 1-10 were constructed and evaluated in pro-megakaryocytes (Figure 12), megakaryocytes (Figure 13), and platelets (Figure 14).
[0238] It was determined that optimal vectors could include various components of one or more of enhancer regions HS3, E2, E9, and E10. It was noted that the HS3 is 531bp, E2 is 3678bp, E9 is 555 bp, and E10 is 455bp. Particularly in view of the length of enhancer region E2, it was desirable to identified smaller effective fragments of these regions. An analysis of enhancer element 2 (260kb downstream of WAS) was found to contain 5 fragments approximating about 3.7 kb. The 5 E2 fragments: E2-1 (first half of core sub element 1 + second half of core sub-element 1, see, e.g., Table 1, SEQ ID NOs:3 and 4), respectively), E2-2 (see, e.g., Table 1, SEQ ID NO:5) , E2-3 (see, e.g., Table 1, SEQ ID NO:6), E2-4 (see, e.g., Table 1, SEQ ID NO:7), and E2-5 (see, e.g., Table 1, SEQ ID NO:8) were cloned into 5 different vectors so that the most active fragments of E2 could be identified for inclusion in the vector. The expression of these vectors in in pro megakaryocytes (Figure 15), megakaryocytes (Figure 16), and platelets (Figure 17) was determined. As shown in Figures 15-17, no one element of E2 seems to be the main driver of expression. Sub-elements 2 and 3 of E2 seem not to have any effect on expression when tested alone. E2 is a driver of megakaryocyte expression but desirably can be slimmed down. This enhancer provides 3 fold higher expression than WAS 1.6 in platelets, and 1.6 fold higher expression than WAS 1.6 in megakaryocytes. However, the E2 enhancer under expresses compared to the g-retroviral vector, but the increase compared to WAS 1.6 may be sufficient to increase platelet counts to normal levels. There is a discrepancy of expression of enhancer elements between CB CD34+ differentiated megakaryocytes and megakaryocyte cell line (MEG-01 cells) as elements 1, 2, 3, 7, 8 and 9 each independently drive expression higher than the previous clinical g-retroviral CMMP-mCit vector in MEG-01 cells, but all under express compare to CMMP-mCit in CB CD34+ megakaryocytes.
[0239] In view of these observations certain particular suitable vectors include, but are not limited to:
1) E2(all slim)-HSlpro-mCit-WPRE (5.6kb);
2) E9(slim)-HS3(slim)-E2(all slim)-HSlpro-mCit-WPRE (6.1kb);
3) E9(slim)-HS3(slim)-l,4,5(slim)of E2-HSlpro-mCit-WPRE (5.6kb); and
4) E9(slim)-HS3(slim)-lst half of l(slim) and 5(slim) of E2-HS lpro-mCit- WPRE (5.0kb).
[0240] The sizes listed above are with the mCit reporter in the open reading frame.
Sizes will differ with a nucleic acid encoding Wasp in the open reading frame.
[0241] It is noted that vector (3) above eliminates sub-elements 2 and 3 in Enhancer element 2, while vector (4) above eliminates the second half of sub-element 1, and sub elements 2, 3, and 4 of Enhancer element 2.
[0242] It will also be recognized that for clinical use (e.g., to treat WAS) the mCitrine open reading frame will be replaced with a nucleic acid encoding the WASp protein. Illustrative nucleic acids include, but are not limited to a WAS cDNA, and a codon-optimized WAS nucleic acid.
Example 2
Identification of Lead Candidate Vector
[0243] Example 1, above, described the generation of four lead candidate vectors: 1)
E2(all slim)-HSlpro-mCit-WRPE; 2) E9(slim)-HS3(slim)-E2(all slim)-HSlpro-mCit-WRPE; 3) E9(slim)-HS3(slim)-l,4,5(slim) of E2-HSlpro-mCit-WRPE; and 4) E9(slim)-HS3(slim)-
1st half of 1 (slim) and 5 (slim) of E2-HSlpro-mCit-WRPE. [0244] This example describes the screening of these vectors in CB CD34+ HSPC differentiation megakaryocytes and platelets to determine a lead candidate vector. Additionally, codon optimization of WASp to replace the open reading frame of mCitrine with WASp and to express the actual therapeutic protein was evaluated. Correction of WASp expression in WAS patient T and B cell lines is also demonstrated.
[0245] In order to determine a lead candidate vector, we screened the 4 candidate vectors in megakaryocytes and platelets differentiated from healthy donor (HD) cord blood (CB) CD34+ hematopoietic stem and progenitor cells (HPSCs). Single element vectors containing modified boundaries and fragments of the E2, E9 and HS3 elements are also included to determine if the modified (smaller sized elements) used to create the lead candidates still retain expression of the parental elements. WAS 1.6 (the current WAS vector undergoing clinical trials) and the previously used g-retroviral vector are also included for comparison of expression.
[0246] As shown in Figure 22 the modified ’’slim” versions of E9, HS3 and E2 retain expression of the larger parental elements. Additionally, one lead candidate vector, E9(slim)- HS3(slim)-l,4,5(slim) of E2-HSlpro-mCit-WRPE, has similar level of expression compare to the y-retro viral vector and a 1.8-fold higher expression than WAS 1.6 in CB CD34+ HSPC differentiated megakaryocytes (Figure 22).
[0247] A similar result was seen in the CB CD34+ differentiated platelets as retroviral vector and 1.5 fold higher than WAS 1.6 {see, e.g., Figure 23).
[0248] In view of these data, one lead candidate vector is E9(slim)-HS3(slim)- l,4,5(slim) of E2-HSlpro-mCit-WPRE (WasVec) which:
[0249] 1) Expresses equal/higher than the y-retro in CB CD34+ megakaryocytes;
[0250] 2) Expresses 1.8 fold greater than WAS 1.6;
[0251] 5.6kb w/ mCitrine in the open reading frame;
[0252] 6.4kb w/ WASp in the open reading frame; and
[0253] 3) Expresses 1.5 fold greater than WAS 1.6 in “platelets”.
[0254] In order to screen for the ideal codon optimized sequence of WASp to further improve expression, we immortalized T and B cell lines from WAS patient and used the cell lines to screen different codon optimized versions of the WASp open reading frame in WASVec - (E9(slim)-HS3(slim)-l,4,5(slim) of E2-HS lpro-WASp-WPRE) [0255] The WAS B-cell and T-cell lines were transduced with different versions of
WASVec, each encoding a different codon optimized version of the WASp open reading frame as follows:
[0256] cDNA (native cDNA sequence, unmodified as a control);
[0257] jCAT codon optimization;
[0258] GeneArt codon optimization;
[0259] IDT codon optimization; and
[0260] Benchling codon optimization.
[0261] As shown in Figures 24, and 25, jCAT codon optimization of WASP in
WASVec led to a 1.29 fold and 1.48 fold increase in expression in B cells (Figure 24) and T cells (Figure 25) respectively compared to the non-codon optimized native cDNA. Figures 24, and 25 also show that GeneArt codon optimization led to a 1.13 fold and 1.20 fold increase in expression in B and T cells respectively compare to the native cDNA, while both IDT and Benchling codon optimizations led to a decrease in expression in B-cells and T- cells. None of the codon optimizations seem to significantly affect titer. Additionally, CAT and GeneArt may slightly increase titer (Figure 26).
[0262] In view of these observations, one lead candidate vector is E9(slim)-
HS3(slim)-E2(l,4,5 slim)-HSlpro-WASP(jCAT codon optimized)-WPRE, aka WASVec. This vector is 6.4kb with WASp in the open reading frame
[0263] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
SEQUENCE LISTING
SEO ID NO:l _ HSlpro (minimal endogenous promoter of the WAS gene)
TCAGCCTCAGGCTACCTAGGTGCTTTAGAAAGGAGGCCACCCAGGCCCATGACT
ACTCCTTGCCACAGGGAGCCCTGCACACAGATGTGCTAAGCTCTCGCTGCCAGCC
AGAGGGAGGAGGGTCTGAGCCAGTCAGAAGGAGATGGGCCCCAGAGAGTAAGA
AAGGGGGAGGAGGACCCAAGCTGATCCAAAAGGTGGGTCTAAGCAGTCAAGTG
GAGGAGGGTTCCAATCTGATGGCGGAGGGCCCAAGCTCAGCCTAACGAGGAGGC
CAGGCCCACCAAGGGGCCCCTGGAGGACTTGTTTCCCTTGTCCCTTGTGGTTTTTT
GCATTTCCTGTTCCCTTGCTGCTCATTGCGGAAGTTCCTCTTCTTACCCTGCACCC
AGAGCCTCGCCAGAGAAGACAAGGGCAGAAAGCACC
SEO ID NO:2 Enhancer element 2 (Slim)
GACCTGAAGGGTAAGGGGGTGTGGAGGTTGTGAAGGCGGGAAGGGGGGTAGCC
CCTTCACCAATGTAAACAAGGATGTGGGTTCTGCGGCCACACTCTCCCCCGCCC
TCCCCAGCGGCATTTCCAGCAAGTCACATGTCCTGCGCACAGGCTGGGGGCCCC
CTGCTGCTGCCTTTCTTAGAAGCCAGGACCACAGAGCCCGCACAGTGAGCTACT
TGGGGAGCTATTTCTGTAGACTGAGCTTGGGGTACTGGGAGGCAGTGCTGTTGG
CGATCTGGGGGAAAACCCCCTGAGGGTCCCATTTCTCTGCCACTGACCCCTTTGC
CTGGCAGAAAGGCTCTGCTGCCTCCTAGCTGTGTAGCAATGGATAAGGCCTTTA
ACCTCTCTAAGCTGAAATGTCCTCATGCATAAGGTGGAGACAGTAACTCCTCTT
CCATCTGCTGGGTGTTGTCAAAACTACACAGGAGGTTTGCCAAGCACCTAAGGC
ACAGTAGGTGGTCAAGAAACAGGAACACAATTTGTCATGACGACAATGATTCCT
TCATATGCTAGTTACCCACGAGTCAGCCAGAATGTTGCCATAACCACTTATGAA
GCCCTTCCTCATTTCTGTTGCAATTCCTCAGCCAGAGGAAAGCCCCACCACCTCT
GTCCTCCAGAAAGCCCTCCTCTTCCCAGAAGGCTGGCACACAGGGCTGGGGGCC
AGGCTTGACTTCCCTGTGGAGGGGTCTGAGGTTGGGGGGATGACCTGCAGAGGT
AGGGGAATTCAGAGAACTTGTTTTGATAGAAACTGAAACTCCTCCTCTTGCTGA
CGCTGCACTTGTGGGCAATCTGCACCTGTTCTCCCCTCCCAGAGGCCAGCTGGA
AGGGGGTCCCCCACCATTCTACCTCTGTTCCCATCTCAAACAGGGCTCAGAAAT
CCTCAGAGAGCCTGGATTATCTTCCTATAGGGTCCTACCATTTGCTTTAGAGACA
TCTGAGAACTGCCTACTCCCCAGGTAGCTGCCTGCCTCCCAGAAGAGCCACAGC
AAGTTCCGCTAAGGGCAAAAAAGGAAGCTAGGTGTCTACAGGGAACCTAAAAA
CAAACCACACTAACGTGTGTACACACAGCTGCAGAGGGAGACAGGGACCTGGT
CTGCTCACAGCCCTTGCCACTCCCCCATGGCCCATGTAGAAACAACCATGTGGC
CTTCACTCTGCCCACAGAAGCTAGCACCAGGACCCTGGTCAGGGTTAGAGGTTT
CTGCTGAGTCAAAGCCACATGGAGGGAGGGAGCAAGGGAGAGATGCAGAGTCA
TGTTTCCAGGAGGAGGTTATCTGAGCATAACAGGGACAGGGTGGGCCACAGGA
TACCTCTGAGGCTCAGGTTCCCACCTCCACTCCACCCTCCAACTCTCTGCTCCAC
GTTCCTTCCACTATTCAACACATGGCAGGAAGTCAGCCTGTAGGGCTTCACACT
ACAGTCTAAGCTGACTGCCATCTGTCCCCATCCAGGCGAGGCTGGAAACGGGGG
CAGCAAAAAGCCCAGGGACAAAGTCCCCTTCCCAGGCACATGTATTATGCACTC
TGCACCAAGGAAACCTCCAGATAAGGTCTAGAGACCAAGGGCCATGTACTTCG
GGGGAGAGGCCAGAGGACTTCTGAGGTTTTACAGAGAAAAGCCAAAGGCAGCC
CAGTCAGGGGAAATGTGTAGCCATAGTGCCGATAAGGAAAGGCCTTCAACCTG
CCCTGGTCAGCTCTTCCTGTAAGTAGAGGCCCCTTACCGAAGGCCCCAGTGGGA
GGAAGGGTCGGGAGTA SEO ID NO: 3 1st half of Core Sub-Element 1 of Enhancer Element 2
GACCTGAAGGGTAAGGGGGTGTGGAGGTTGTGAAGGCGGGAAGGGGGGTAGCC
CCTTCACCAATGTAAACAAGGATGTGGGTTCTGCGGCCACACTCTCCCCCGCCC
TCCCCAGCGGCATTTCCAGCAAGTCACATGTCCTGCGCACAGGCTGGGGGCCCC
CTGCTGCTGCCTTTCTTAGAAGCCAGGACCACAGAGCCCGCACAGTGAGCTACT
TGGGGAGCTATTTCTGTAGACTGAGCTTGGGGTACTGGGAGGCAGTGCTGTTGG
C
SEO ID NO:4 2nd half of Core Sub-Element 1 of Enhancer Element 2
GATCTGGGGGAAAACCCCCTGAGGGTCCCATTTCTCTGCCACTGACCCCTTTGCC
TGGCAGAAAGGCTCTGCTGCCTCCTAGCTGTGTAGCAATGGATAAGGCCTTTAA
CCTCTCTAAGCTGAAATGTCCTCATGCATAAGGTGGAGACAGTAACTCCTCTTCC
ATCTGCTGGGTGTTGTCAAAACTACACAGGAGGTTTGCCAAGCACCTAAGGCAC
AGTAGGTGGTCAAGAAACAGGAACACAATTTGTCATGACGACAATGATTCCTTC
ATATGCTAGTTACCCACGAGTCAGCCAGAATGTTGCCATAACCACTTATGAAGC
CCTTCCTCATTTCTGTTGCAATTCCTCAGCCAGAGGAAAGCCCCA
SEO ID NO:5 Core Sub-Element 2 of Enhancer Element 2
CCACCTCTGTCCTCCAGAAAGCCCTCCTCTTCCCAGAAGGCTGGCACACAGGGC
TGGGGGCCAGGCTTGACTTCCCTGTGGAGGGGTCTGAGGTTGGGGGGATGACCT
GCAGAGGTAGGGGAATTCAGAGAACTTGTTTTGATAGAAACTGAAACTCCTCCT
CTTGCTGACGCTGCACTTGTGGGCAATCTGCACCTGTTCTCCCCTCCCAGAGGCC
AGCTGGAAGGGGGTCCCCCACCATTCTACCTCTGTTCCCATCTCAAACAGGGCT
CAGAAATCCTCA
SEO ID NO:6 Core Sub-Element 3 of Enhancer Element 2
GAGAGCCTGGATTATCTTCCTATAGGGTCCTACCATTTGCTTTAGAGACATCTGA
GAACTGCCTACTCCCCAGGTAGCTGCCTGCCTCCCAGAAGAGCCACAGCAAGTT
CCGCTAAGGGCAAAAAAGGAAGCTAGGTGTCTACAGGGAACCTAAAAACAAAC
CACACTAACGTGTGTACACACAGCTGCAGAGGGAGACAGGGACCTGGTCTGCT
SEO ID NO:7 Core-Sub Element 4 of Enhancer Element 2
CACAGCCCTTGCCACTCCCCCATGGCCCATGTAGAAACAACCATGTGGCCTTCA
CTCTGCCCACAGAAGCTAGCACCAGGACCCTGGTCAGGGTTAGAGGTTTCTGCT
GAGTCAAAGCCACATGGAGGGAGGGAGCAAGGGAGAGATGCAGAGTCATGTTT
CCAGGAGGAGGTTATCTGAGCATAACAGGGACAGGGTGGGCCACAGGATACCT
CTGAGGCTCAGGTTCCCACCTCCACTCCACC
SEO ID NO: 8 Core-Sub Element 5 of Enhancer Element 2
CTCCAACTCTCTGCTCCACGTTCCTTCCACTATTCAACACATGGCAGGAAGTCAG
CCTGTAGGGCTTCACACTACAGTCTAAGCTGACTGCCATCTGTCCCCATCCAGGC
GAGGCTGGAAACGGGGGCAGCAAAAAGCCCAGGGACAAAGTCCCCTTCCCAGG
CACATGTATTATGCACTCTGCACCAAGGAAACCTCCAGATAAGGTCTAGAGACC
AAGGGCCATGTACTTCGGGGGAGAGGCCAGAGGACTTCTGAGGTTTTACAGAG
AAAAGCCAAAGGCAGCCCAGTCAGGGGAAATGTGTAGCCATAGTGCCGATAAG
GAAAGGCCTTCAACCTGCCCTGGTCAGCTCTTCCTGTAAGTAGAGGCCCCTTAC
CGAAGGCCCCAGTGGGAGGAAGGGTCGGGAGTA SEO ID NO:9Enhancer element HS3 (full)
CTAAAAGCCACCAAAACCAAGACAGCAATGAAAGTAACCTCTGGTCTTCCT
AACTGCTCATTATACACTAATTATGATGCACTAGCATGCTAAGAGACACTTC
CACCAGCGCCATGACAGTGCCATGGCAACATCAAGAAGTCACCCCACATGG
TCTAAAAAGGGGAAGAACCTTCAGTTCCCGGAATTGCCCACCCCTTTCCTG
GAAAACTCATGAATAATCCACCCCTTGTTTAGCATATAATTAAGAAATAACT
GTAAGTATCCTTAGTCCAGCAGCCCAAGCTGCTGCTCTGCCTATGGAGTAG
CCATTCTTTATTCCTTTACTTTCTTAATAAACTTGCTTTCACATTAAAAACAA
CAACAACAACACCAACAACCAGCAAATATATCAGAGGCTTAGCATCCCCTA
TTCAAGGGGACAGTTCTGGAGGCCGAGAAAGAAGTAGAAAGGTGGAGAAT
AAAAGACACAAAGGAGCAGAAGAATTTTATCTTGGCCTCCATGTGGCACCT
CACAGCTTGGGAACAACA
SEO ID NQ:10 _ enhancer element HS3 core
CTAAAAGCCACCAAAACCAAGACAGCAATGAAAGTAACCTCTGGTCTTCCTAAC
TGCTCATTATACACTAATTATGATGCACTAGCATGCTAAGAGACACTTCCACCA
GCGCCATGACAGTGCCATGGCAACATCAAGAAGTCACCCCACATGGTCTAAAA
AGGGGAAGAACCTTCAGTTCCCGGAATTGCCCACCCCTTTCCTGGAAAACTCAT
GAATAATCCACCCCTTGTTTAGCATATAAT
SEO ID NO:ll _ Enhancer element E9 (full)
GGGATCTGTGTGCAGATTTACCTCTACCTACACCTGGCTGGGGATCATAAAGAA
AATCAAGGGATGCGCTACCTCCATGAATCCAGGTTTCAGCAGAGCTAAGGGAGT
GAAATTTGGGGCCATTTTACTTGTCTCAAACCTTGTTATCTGAGAGAAAGCTAG
AGCTTCCTTCTTTCAGCCCCCAGAGACAATGTGGCCAGGCTCCGGAGGGCTGGG
AAGATGAGCAATGCTTGTGAGTCACCATGATAGGAAGCAGAAGGGTCAGGAAG
TCCCTGGGAGCAAGGCTTAGGGTTAGGGTTAGGGAGAAAACACTTGGGCCTGG
AGGCTCGGGGCAGGCTTCCTAGAGGGGAGGGGTAGGAAGAGGCAGTGACAGGG
CCCACAGCAATGGAGAGGAGTTGGACTGCAGGGATGGGGTAGGGGGACAGACG
ACAAGGGACACTCAGAAGACTAATGTCTGGGAGTGGGAAAACAGTGTTTGCTC
AGCCAGGAAGCTGCATCCAGCTCTTTTATCATTTGTAGAAGACCAAGTACCCAG
GCCTGGGAGGAGCCCAGAGA
SEO ID NO:12 _ Enhancer element E9 core
CAAACCTTGTTATCTGAGAGAAAGCTAGAGCTTCCTTCTTTCAGCCCCCAGAGA
CAATGTGGCCAGGCTCCGGAGGGCTGGGAAGATGAGCAATGCTTGTGAGTCAC
CATGATAGGAAGCAGAAGGGTCAGGAAGTCCCTGGGAGCAAGGCTTAGGGTTA
GGGTTAGGGAGAAAACACTTGGGCCTGGAGGCTCGGGGCAGGCTTCCTAGAGG
GGAGGGGTA
SEO ID NO:13 _ WAS cDNA atgagtgggggcccaatgggaggaaggcccgggggccgaggagcaccagcggttcagcagaacataccctccaccctcctccag gaccacgagaaccagcgactctttgagatgcttggacgaaaatgcttgacgctggccactgcagttgttcagctgtacctggcgctgcc ccctggagctgagcactggaccaaggagcattgtggggctgtgtgcttcgtgaaggataacccccagaagtcctacttcatccgccttt acggccttcaggctggtcggctgctctgggaacaggagctgtactcacagcttgtctactccacccccacccccttcttccacaccttcg ctggagatgactgccaagcggggctgaactttgcagacgaggacgaggcccaggccttccgggccctcgtgcaggagaagataca aaaaaggaatcagaggcaaagtggagacagacgccagctacccccaccaccaacaccagccaatgaagagagaagaggagggc tcccacccctgcccctgcatccaggtggagaccaaggaggccctccagtgggtccgctctccctggggctggcgacagtggacatc cagaaccctgacatcacgagttcacgataccgtgggctcccagcacctggacctagcccagctgataagaaacgctcagggaagaa gaagatcagcaaagctgatattggtgcacccagtggattcaagcatgtcagccacgtggggtgggacccccagaatggatttgacgt gaacaacctcgacccagatctgcggagtctgttctccagggcaggaatcagcgaggcccagctcaccgacgccgagacctctaaac ttatctacgacttcattgaggaccagggtgggctggaggctgtgcggcaggagatgaggcgccaggagccacttccgccgccccca ccgccatctcgaggagggaaccagctcccccggccccctattgtggggggtaacaagggtcgttctggtccactgccccctgtacctt tggggattgccccacccccaccaacaccccggggacccccacccccaggccgagggggccctccaccaccaccccctccagcta ctggacgttctggaccactgccccctccaccccctggagctggtgggccacccatgccaccaccaccgccaccaccgccaccgccg cccagctccgggaatggaccagcccctcccccactccctcctgctctggtgcctgccgggggcctggcccctggtgggggtcgggg agcgcttttggatcaaatccggcagggaattcagctgaacaagacccctggggccccagagagctcagcgctgcagccaccacctc agagctcagagggactggtgggggccctgatgcacgtgatgcagaagagaagcagagccatccactcctccgacgaaggggagg accaggctggcgatgaagatgaagatgatgaatgggatgactga
SEO ID NO:14 Codon optimized WAS
ATGAGCGGCGGCCCCATGGGCGGCCGCCCCGGCGGCCGCGGCGCCCCCGCCGTG
CAGCAGAACATCCCCAGCACCCTGCTGCAGGACCACGAGAACCAGCGCCTGTTC
GAGATGCTGGGCCGCAAGTGCCTGACCCTGGCCACCGCCGTGGTGCAGCTGTAC
CTGGCCCTGCCCCCCGGCGCCGAGCACTGGACCAAGGAGCACTGCGGCGCCGTG
TGCTTCGTGAAGGACAACCCCCAGAAGAGCTACTTCATCCGCCTGTACGGCCTGC
AGGCCGGCCGCCTGCTGTGGGAGCAGGAGCTGTACAGCCAGCTGGTGTACAGCA
CCCCCACCCCCTTCTTCCACACCTTCGCCGGCGACGACTGCCAGGCCGGCCTGAA
CTTCGCCGACGAGGACGAGGCCCAGGCCTTCCGCGCCCTGGTGCAGGAGAAGAT
CCAGAAGCGCAACCAGCGCCAGAGCGGCGACCGCCGCCAGCTGCCCCCCCCCCC
CACCCCCGCCAACGAGGAGCGCCGCGGCGGCCTGCCCCCCCTGCCCCTGCACCCC
GGCGGCGACCAGGGCGGCCCCCCCGTGGGCCCCCTGAGCCTGGGCCTGGCCACC
GTGGACATCCAGAACCCCGACATCACCAGCAGCCGCTACCGCGGCCTGCCCGCC
CCCGGCCCCAGCCCCGCCGACAAGAAGCGCAGCGGCAAGAAGAAGATCAGCAA
GGCCGACATCGGCGCCCCCAGCGGCTTCAAGCACGTGAGCCACGTGGGCTGGGA
CCCCCAGAACGGCTTCGACGTGAACAACCTGGACCCCGACCTGCGCAGCCTGTTC
AGCCGCGCCGGCATCAGCGAGGCCCAGCTGACCGACGCCGAGACCAGCAAGCTG
ATCTACGACTTCATCGAGGACCAGGGCGGCCTGGAGGCCGTGCGCCAGGAGATG
CGCCGCCAGGAGCCCCTGCCCCCCCCCCCCCCCCCCAGCCGCGGCGGCAACCAG
CTGCCCCGCCCCCCCATCGTGGGCGGCAACAAGGGCCGCAGCGGCCCCCTGCCC
CCCGTGCCCCTGGGCATCGCCCCCCCCCCCCCCACCCCCCGCGGCCCCCCCCCCC
CCGGCCGCGGCGGCCCCCCCCCCCCCCCCCCCCCCGCCACCGGCCGCAGCGGCCC
CCTGCCCCCCCCCCCCCCCGGCGCCGGCGGCCCCCCCATGCCCCCCCCCCCCCCC
CCCCCCCCCCCCCCCCCCAGCAGCGGCAACGGCCCCGCCCCCCCCCCCCTGCCCC
CCGCCCTGGTGCCCGCCGGCGGCCTGGCCCCCGGCGGCGGCCGCGGCGCCCTGCT
GGACCAGATCCGCCAGGGCATCCAGCTGAACAAGACCCCCGGCGCCCCCGAGAG
CAGCGCCCTGCAGCCCCCCCCCCAGAGCAGCGAGGGCCTGGTGGGCGCCCTGAT
GCACGTGATGCAGAAGCGCAGCCGCGCCATCCACAGCAGCGACGAGGGCGAGG
ACCAGGCCGGCGACGAGGACGAGGACGACGAGTGGGACGACTAA
SEP ID NO : 15 E2(all slimVHS Ipro-mCit- WPRE
Sequence from CMV (red arrow in Figure 18) going clockwise:
AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG
ACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAG
GGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGG
CAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACG
GTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTAC TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGG
CAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC
CACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTT
CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTA
CGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGGTCTCTCTGGT
TAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTA
AGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTG
TGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCT
AGCagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagc gcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggt gcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataa attaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaa atactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcat caaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagca agcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattga accattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttcctt gggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtg cagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggc aagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgct gtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaaca attacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgg gcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaat agtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggaccc gacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtat cggttaacttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaac taaagaattacaaaaacaaattacaaaaattcaaaattttatcgatcacgagactagcctcgagGACCTGAAGGGTAAG
GGGGTGTGGAGGTTGTGAAGGCGGGAAGGGGGGTAGCCCCTTCACCAATGTAA
ACAAGGATGTGGGTTCTGCGGCCACACTCTCCCCCGCCCTCCCCAGCGGCATTT
CCAGCAAGTCACATGTCCTGCGCACAGGCTGGGGGCCCCCTGCTGCTGCCTTTC
TTAGAAGCCAGGACCACAGAGCCCGCACAGTGAGCTACTTGGGGAGCTATTTCT
GTAGACTGAGCTTGGGGTACTGGGAGGCAGTGCTGTTGGCGATCTGGGGGAAA
ACCCCCTGAGGGTCCCATTTCTCTGCCACTGACCCCTTTGCCTGGCAGAAAGGCT
CTGCTGCCTCCTAGCTGTGTAGCAATGGATAAGGCCTTTAACCTCTCTAAGCTGA
AATGTCCTCATGCATAAGGTGGAGACAGTAACTCCTCTTCCATCTGCTGGGTGTT
GTCAAAACTACACAGGAGGTTTGCCAAGCACCTAAGGCACAGTAGGTGGTCAA
GAAACAGGAACACAATTTGTCATGACGACAATGATTCCTTCATATGCTAGTTAC
CCACGAGTCAGCCAGAATGTTGCCATAACCACTTATGAAGCCCTTCCTCATTTCT
GTTGCAATTCCTCAGCCAGAGGAAAGCCCCACCACCTCTGTCCTCCAGAAAGCC
CTCCTCTTCCCAGAAGGCTGGCACACAGGGCTGGGGGCCAGGCTTGACTTCCCT
GTGGAGGGGTCTGAGGTTGGGGGGATGACCTGCAGAGGTAGGGGAATTCAGAG
AACTTGTTTTGATAGAAACTGAAACTCCTCCTCTTGCTGACGCTGCACTTGTGGG
CAATCTGCACCTGTTCTCCCCTCCCAGAGGCCAGCTGGAAGGGGGTCCCCCACC
ATTCTACCTCTGTTCCCATCTCAAACAGGGCTCAGAAATCCTCAGAGAGCCTGG
ATTATCTTCCTATAGGGTCCTACCATTTGCTTTAGAGACATCTGAGAACTGCCTA
CTCCCCAGGTAGCTGCCTGCCTCCCAGAAGAGCCACAGCAAGTTCCGCTAAGGG
CAAAAAAGGAAGCTAGGTGTCTACAGGGAACCTAAAAACAAACCACACTAACG
TGTGTACACACAGCTGCAGAGGGAGACAGGGACCTGGTCTGCTCACAGCCCTTG
CCACTCCCCCATGGCCCATGTAGAAACAACCATGTGGCCTTCACTCTGCCCACA
GAAGCTAGCACCAGGACCCTGGTCAGGGTTAGAGGTTTCTGCTGAGTCAAAGCC
ACATGGAGGGAGGGAGCAAGGGAGAGATGCAGAGTCATGTTTCCAGGAGGAGG
TTATCTGAGCATAACAGGGACAGGGTGGGCCACAGGATACCTCTGAGGCTCAG
GTTCCCACCTCCACTCCACCCTCCAACTCTCTGCTCCACGTTCCTTCCACTATTCA ACACATGGCAGGAAGTCAGCCTGTAGGGCTTCACACTACAGTCTAAGCTGACTG
CCATCTGTCCCCATCCAGGCGAGGCTGGAAACGGGGGCAGCAAAAAGCCCAGG
GACAAAGTCCCCTTCCCAGGCACATGTATTATGCACTCTGCACCAAGGAAACCT
CCAGATAAGGTCTAGAGACCAAGGGCCATGTACTTCGGGGGAGAGGCCAGAGG
ACTTCTGAGGTTTTACAGAGAAAAGCCAAAGGCAGCCCAGTCAGGGGAAATGT
GTAGCCATAGTGCCGATAAGGAAAGGCCTTCAACCTGCCCTGGTCAGCTCTTCC
TGTAAGTAGAGGCCCCTTACCGAAGGCCCCAGTGGGAGGAAGGGTCGGGAGTA ctcgagTCAGCCTCAGGCTACCTAGGTGCTTTAGAAAGGAGGCCACCCAGGCCCAT
GACTACTCCTTGCCACAGGGAGCCCTGCACACAGATGTGCTAAGCTCTCGCTGC
CAGCCAGAGGGAGGAGGGTCTGAGCCAGTCAGAAGGAGATGGGCCCCAGAGA
GTAAGAAAGGGGGAGGAGGACCCAAGCTGATCCAAAAGGTGGGTCTAAGCAGT
CAAGTGGAGGAGGGTTCCAATCTGATGGCGGAGGGCCCAAGCTCAGCCTAACG
AGGAGGCCAGGCCCACCAAGGGGCCCCTGGAGGACTTGTTTCCCTTGTCCCTTG
TGGTTTTTTGCATTTCCTGTTCCCTTGCTGCTCATTGCGGAAGTTCCTCTTCTTAC
CCTGCACCCAGAGCCTCGCCAGAGAAGACAAGGGCAGAAAGCACCATGgtgagcaa gggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccgg cgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccac cctcgtgaccaccttcggctacggcctgatgtgcttcgcccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatg cccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggc gacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaac tacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgagg acggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacc tgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggat cactctcggcatggacgagctgtacaagtgactgcaggaattcgagcatcttaccgccatttattcccatatttgttctgtttttcttgatttg ggtatacatttaaatgttaataaaacaaaatggtggggcaatcatttacatttttagggatatgtaattactagttcaggtgtattgccacaa gacaaacatgttaagaaactttcccgttatttacgctctgttcctgttaatcaacctctggattacaaaatttgtgaaagattgactgatattc ttaactatgttgctccttttacgctgtgtggatatgctgctttaatgcctctgtatcatgctattgcttcccgtacggctttcgttttctcctcctt gtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtccgtcaacgtggcgtggtgtgctctgtgtttgctgacgcaacc cccactggctggggcattgccaccacctgtcaactcctttctgggactttcgctttccccctcccgatcgccacggcagaactcatcgc cgcctgccttgcccgctgctggacaggggctaggttgctgggcactgataattccgtggtgttgtcggggaagggcctgctgccgg ctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattcgagctcgg tacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcc caacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaac ctactgcttaagcctcaataaagcttgccttgagtgcttCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTC
TGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCagta gtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatgg ttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatctt atcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggct gactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctag ggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggc gttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaa cagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtg accgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctc taaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtg ggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacact caaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaac gcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatac attcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatagcacctagatcaagagacaggatgaggatcgtttc gcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagaca atcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctga atgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaa gcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatc atggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagc acgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgcca ggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggc cgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaaga gcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttga cgagttcttctgaattattaacgcttacaatttcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatcaggtggca cttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgaccaaaatcccttaacgtg agttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaa acaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagag cgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgc taatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgca gcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtga gctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgc acgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgc tcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgtt ctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagc gcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgca gctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccag gctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacg ccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttggccattgcatacgttgtatccatatcataatat gtacatttatattggctcatgtccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttc atagcccatatatgg
SEP ID NO:16 E9(slim)-HS3(slim)-E2(all slim)-HSlpro-mCit-WPRE (added the
E9(slim) and HS3(slim) elements)
Sequence from CMV (red arrow) going clockwise in Figure 19:
AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGA
CCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGG
ACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAG
TACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAA
ATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGG
CAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGT
ACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCC
CATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAA
TGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGG
GAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGGTCTCTCTGGTTAGACCA
GATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAA
TAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGG
TAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCagtggcgcc cgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagag gcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtat taagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatg ggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctaca accatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaa gacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcaga cctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcaccca ccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcagga agcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctga gggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagat acctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagta ataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaa ttgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataa caaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaataga gttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaaga aggtggagagagagaeagagaeagateeattegattagtgaaeggatetegaeggtateggttaaettttaaaagaaaaggggggatt ggggggtaeagtgeaggggaaagaatagtagaeataatageaaeagaeataeaaaetaaagaattaeaaaaaeaaattaeaaaaatt caaaattttatcgatcacgagactagcctcgagCAAACCTTGTTATCTGAGAGAAAGCTAGAGCTTCC
TTCTTTCAGCCCCCAGAGACAATGTGGCCAGGCTCCGGAGGGCTGGGAAGATGA
GCAATGCTTGTGAGTCACCATGATAGGAAGCAGAAGGGTCAGGAAGTCCCTGGG
AGCAAGGCTTAGGGTTAGGGTTAGGGAGAAAACACTTGGGCCTGGAGGCTCGGG
GCAGGCTTCCTAGAGGGGAGGGGTACTAAAAGCCACCAAAACCAAGACAGCAAT
GAAAGTAACCTCTGGTCTTCCTAACTGCTCATTATACACTAATTATGATGCACTA
GCATGCTAAGAGACACTTCCACCAGCGCCATGACAGTGCCATGGCAACATCAAG
AAGTCACCCCACATGGTCTAAAAAGGGGAAGAACCTTCAGTTCCCGGAATTGCC
CACCCCTTTCCTGGAAAACTCATGAATAATCCACCCCTTGTTTAGCATATAATGA
CCTGAAGGGTAAGGGGGTGTGGAGGTTGTGAAGGCGGGAAGGGGGGTAGCCCCT
TCACCAATGTAAACAAGGATGTGGGTTCTGCGGCCACACTCTCCCCCGCCCTCCC
CAGCGGCATTTCCAGCAAGTCACATGTCCTGCGCACAGGCTGGGGGCCCCCTGCT
GCTGCCTTTCTTAGAAGCCAGGACCACAGAGCCCGCACAGTGAGCTACTTGGGG
AGCTATTTCTGTAGACTGAGCTTGGGGTACTGGGAGGCAGTGCTGTTGGCGATCT
GGGGGAAAACCCCCTGAGGGTCCCATTTCTCTGCCACTGACCCCTTTGCCTGGCA
GAAAGGCTCTGCTGCCTCCTAGCTGTGTAGCAATGGATAAGGCCTTTAACCTCTC
TAAGCTGAAATGTCCTCATGCATAAGGTGGAGACAGTAACTCCTCTTCCATCTGC
TGGGTGTTGTCAAAACTACACAGGAGGTTTGCCAAGCACCTAAGGCACAGTAGG
TGGTCAAGAAACAGGAACACAATTTGTCATGACGACAATGATTCCTTCATATGCT
AGTTACCCACGAGTCAGCCAGAATGTTGCCATAACCACTTATGAAGCCCTTCCTC
ATTTCTGTTGCAATTCCTCAGCCAGAGGAAAGCCCCACCACCTCTGTCCTCCAGA
AAGCCCTCCTCTTCCCAGAAGGCTGGCACACAGGGCTGGGGGCCAGGCTTGACTT
CCCTGTGGAGGGGTCTGAGGTTGGGGGGATGACCTGCAGAGGTAGGGGAATTCA
GAGAACTTGTTTTGATAGAAACTGAAACTCCTCCTCTTGCTGACGCTGCACTTGT
GGGCAATCTGCACCTGTTCTCCCCTCCCAGAGGCCAGCTGGAAGGGGGTCCCCCA
CCATTCTACCTCTGTTCCCATCTCAAACAGGGCTCAGAAATCCTCAGAGAGCCTG
GATTATCTTCCTATAGGGTCCTACCATTTGCTTTAGAGACATCTGAGAACTGCCTA
CTCCCCAGGTAGCTGCCTGCCTCCCAGAAGAGCCACAGCAAGTTCCGCTAAGGG
CAAAAAAGGAAGCTAGGTGTCTACAGGGAACCTAAAAACAAACCACACTAACGT
GTGTACACACAGCTGCAGAGGGAGACAGGGACCTGGTCTGCTCACAGCCCTTGC
CACTCCCCCATGGCCCATGTAGAAACAACCATGTGGCCTTCACTCTGCCCACAGA
AGCTAGCACCAGGACCCTGGTCAGGGTTAGAGGTTTCTGCTGAGTCAAAGCCAC
ATGGAGGGAGGGAGCAAGGGAGAGATGCAGAGTCATGTTTCCAGGAGGAGGTT
ATCTGAGCATAACAGGGACAGGGTGGGCCACAGGATACCTCTGAGGCTCAGGTT
CCCACCTCCACTCCACCCTCCAACTCTCTGCTCCACGTTCCTTCCACTATTCAACA
CATGGCAGGAAGTCAGCCTGTAGGGCTTCACACTACAGTCTAAGCTGACTGCCAT
CTGTCCCCATCCAGGCGAGGCTGGAAACGGGGGCAGCAAAAAGCCCAGGGACA
AAGTCCCCTTCCCAGGCACATGTATTATGCACTCTGCACCAAGGAAACCTCCAGA
TAAGGTCTAGAGACCAAGGGCCATGTACTTCGGGGGAGAGGCCAGAGGACTTCT
GAGGTTTTACAGAGAAAAGCCAAAGGCAGCCCAGTCAGGGGAAATGTGTAGCCA
TAGTGCCGATAAGGAAAGGCCTTCAACCTGCCCTGGTCAGCTCTTCCTGTAAGTA
GAGGCCCCTTACCGAAGGCCCCAGTGGGAGGAAGGGTCGGGAGTActcgagTCAGC
CTCAGGCTACCTAGGTGCTTTAGAAAGGAGGCCACCCAGGCCCATGACTACTCCT
TGCCACAGGGAGCCCTGCACACAGATGTGCTAAGCTCTCGCTGCCAGCCAGAGG
GAGGAGGGTCTGAGCCAGTCAGAAGGAGATGGGCCCCAGAGAGTAAGAAAGGG GGAGGAGGACCCAAGCTGATCCAAAAGGTGGGTCTAAGCAGTCAAGTGGAGGA
GGGTTCCAATCTGATGGCGGAGGGCCCAAGCTCAGCCTAACGAGGAGGCCAGGC
CCACCAAGGGGCCCCTGGAGGACTTGTTTCCCTTGTCCCTTGTGGTTTTTTGCATT
TCCTGTTCCCTTGCTGCTCATTGCGGAAGTTCCTCTTCTTACCCTGCACCCAGAGC
CTCGCCAGAGAAGACAAGGGCAGAAAGCACCATGgtgagcaagggcgaggagctgttcaccggg gtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacct acggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcc tgatgtgcttcgcccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgca ccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctg aagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggc cgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactac cagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagacc ccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtga ctgcaggaattcgagcatcttaccgccatttattcccatatttgttctgtttttcttgatttgggtatacatttaaatgttaataaaacaaaatggt ggggcaatcatttacatttttagggatatgtaattactagttcaggtgtattgccacaagacaaacatgttaagaaactttcccgttatttacg ctctgttcctgttaatcaacctctggattacaaaatttgtgaaagattgactgatattcttaactatgttgctccttttacgctgtgtggatatgct gctttaatgcctctgtatcatgctattgcttcccgtacggctttcgttttctcctccttgtataaatcctggttgctgtctctttatgaggagttgt ggcccgttgtccgtcaacgtggcgtggtgtgctctgtgtttgctgacgcaacccccactggctggggcattgccaccacctgtcaactc ctttctgggactttcgctttccccctcccgatcgccacggcagaactcatcgccgcctgccttgcccgctgctggacaggggctaggtt gctgggcactgataattccgtggtgttgtcggggaagggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcaga cgagtcggatctccctttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatct tagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctc tggttagaccagatctgagcctgggagctctctggctaactagggaacctactgcttaagcctcaataaagcttgccttgagtgcttCA
AGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCC
TTTTAGTCAGTGTGGAAAATCTCTAGCagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaa atgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcat ttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatccc gcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctct gagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcac tggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggc gtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgc attaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttc ctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgacc ccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttcttt aatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattg gttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtg cgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatagcacc tagatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggct attcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttg tcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgc gcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcac cttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccacca agcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggc tcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttg ccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgtt ggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagc gcatcgccttctatcgccttcttgacgagttcttctgaattattaacgcttacaatttcctgatgcggtattttctccttacgcatctgtgcggta tttcacaccgcatcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctca tgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgc gcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaagg taactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgc ctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagtt accggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgag atacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcgga acaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtc gatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttt tgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaa cgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattc attaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggc accccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccat gattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttggccattgcatacgttgtatccatatca taatatgtacatttatattggctcatgtccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcatt agttcatagcccatatatgg
SEQ ID NO:17 E9(slim)-HS3(slim)-E2(l,4,5 slim)-HSlpro-mCit-WPRE (Deleted
Core Sub-Element 2 of Element 2 and Core Sub-Element 3 of Element 2)
Sequence from CMV (red arrow) going clockwise in Figure 20
AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGA
CCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGG
ACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAG
TACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAA
ATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGG
CAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGT
ACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCC
CATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAA
TGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGG
GAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGGTCTCTCTGGTTAGACCA
GATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAA
TAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGG
TAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCagtggcgcc cgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagag gcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtat taagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatg ggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctaca accatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaa gacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcaga cctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcaccca ccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcagga agcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctga gggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagat acctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagta ataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaa ttgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataa caaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaataga gttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaaga aggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcggttaacttttaaaagaaaaggggggatt ggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaatt caaaattttatcgatcacgagactagcctcgagCAAACCTTGTTATCTGAGAGAAAGCTAGAGCTTCC
TTCTTTCAGCCCCCAGAGACAATGTGGCCAGGCTCCGGAGGGCTGGGAAGATGA
GCAATGCTTGTGAGTCACCATGATAGGAAGCAGAAGGGTCAGGAAGTCCCTGGG
AGCAAGGCTTAGGGTTAGGGTTAGGGAGAAAACACTTGGGCCTGGAGGCTCGGG GCAGGCTTCCTAGAGGGGAGGGGTACTAAAAGCCACCAAAACCAAGACAGCAAT
GAAAGTAACCTCTGGTCTTCCTAACTGCTCATTATACACTAATTATGATGCACTA
GCATGCTAAGAGACACTTCCACCAGCGCCATGACAGTGCCATGGCAACATCAAG
AAGTCACCCCACATGGTCTAAAAAGGGGAAGAACCTTCAGTTCCCGGAATTGCC
CACCCCTTTCCTGGAAAACTCATGAATAATCCACCCCTTGTTTAGCATATAATGA
CCTGAAGGGTAAGGGGGTGTGGAGGTTGTGAAGGCGGGAAGGGGGGTAGCCCCT
TCACCAATGTAAACAAGGATGTGGGTTCTGCGGCCACACTCTCCCCCGCCCTCCC
CAGCGGCATTTCCAGCAAGTCACATGTCCTGCGCACAGGCTGGGGGCCCCCTGCT
GCTGCCTTTCTTAGAAGCCAGGACCACAGAGCCCGCACAGTGAGCTACTTGGGG
AGCTATTTCTGTAGACTGAGCTTGGGGTACTGGGAGGCAGTGCTGTTGGCGATCT
GGGGGAAAACCCCCTGAGGGTCCCATTTCTCTGCCACTGACCCCTTTGCCTGGCA
GAAAGGCTCTGCTGCCTCCTAGCTGTGTAGCAATGGATAAGGCCTTTAACCTCTC
TAAGCTGAAATGTCCTCATGCATAAGGTGGAGACAGTAACTCCTCTTCCATCTGC
TGGGTGTTGTCAAAACTACACAGGAGGTTTGCCAAGCACCTAAGGCACAGTAGG
TGGTCAAGAAACAGGAACACAATTTGTCATGACGACAATGATTCCTTCATATGCT
AGTTACCCACGAGTCAGCCAGAATGTTGCCATAACCACTTATGAAGCCCTTCCTC
ATTTCTGTTGCAATTCCTCAGCCAGAGGAAAGCCCCACACAGCCCTTGCCACTCC
CCCATGGCCCATGTAGAAACAACCATGTGGCCTTCACTCTGCCCACAGAAGCTAG
CACCAGGACCCTGGTCAGGGTTAGAGGTTTCTGCTGAGTCAAAGCCACATGGAG
GGAGGGAGCAAGGGAGAGATGCAGAGTCATGTTTCCAGGAGGAGGTTATCTGAG
CATAACAGGGACAGGGTGGGCCACAGGATACCTCTGAGGCTCAGGTTCCCACCT
CCACTCCACCCTCCAACTCTCTGCTCCACGTTCCTTCCACTATTCAACACATGGCA
GGAAGTCAGCCTGTAGGGCTTCACACTACAGTCTAAGCTGACTGCCATCTGTCCC
CATCCAGGCGAGGCTGGAAACGGGGGCAGCAAAAAGCCCAGGGACAAAGTCCC
CTTCCCAGGCACATGTATTATGCACTCTGCACCAAGGAAACCTCCAGATAAGGTC
TAGAGACCAAGGGCCATGTACTTCGGGGGAGAGGCCAGAGGACTTCTGAGGTTT
TACAGAGAAAAGCCAAAGGCAGCCCAGTCAGGGGAAATGTGTAGCCATAGTGCC
GATAAGGAAAGGCCTTCAACCTGCCCTGGTCAGCTCTTCCTGTAAGTAGAGGCCC
CTTACCGAAGGCCCCAGTGGGAGGAAGGGTCGGGAGTActcgagTCAGCCTCAGGC
TACCTAGGTGCTTTAGAAAGGAGGCCACCCAGGCCCATGACTACTCCTTGCCACA
GGGAGCCCTGCACACAGATGTGCTAAGCTCTCGCTGCCAGCCAGAGGGAGGAGG
GTCTGAGCCAGTCAGAAGGAGATGGGCCCCAGAGAGTAAGAAAGGGGGAGGAG
GACCCAAGCTGATCCAAAAGGTGGGTCTAAGCAGTCAAGTGGAGGAGGGTTCCA
ATCTGATGGCGGAGGGCCCAAGCTCAGCCTAACGAGGAGGCCAGGCCCACCAAG
GGGCCCCTGGAGGACTTGTTTCCCTTGTCCCTTGTGGTTTTTTGCATTTCCTGTTCC
CTTGCTGCTCATTGCGGAAGTTCCTCTTCTTACCCTGCACCCAGAGCCTCGCCAGA
GAAGACAAGGGCAGAAAGCACCATGgtgagcaagggcgaggagctgttcaccggggtggtgcccatcctg gtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgacc ctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgatgtgcttcgccc gctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaagg acgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgactt caaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaag aacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacaccc ccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcg cgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtgactgcaggaattcga gcatcttaccgccatttattcccatatttgttctgtttttcttgatttgggtatacatttaaatgttaataaaacaaaatggtggggcaatcattta catttttagggatatgtaattactagttcaggtgtattgccacaagacaaacatgttaagaaactttcccgttatttacgctctgttcctgttaat caacctctggattacaaaatttgtgaaagattgactgatattcttaactatgttgctccttttacgctgtgtggatatgctgctttaatgcctctg tatcatgctattgcttcccgtacggctttcgttttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtccgtc aacgtggcgtggtgtgctctgtgtttgctgacgcaacccccactggctggggcattgccaccacctgtcaactcctttctgggactttcg ctttccccctcccgatcgccacggcagaactcatcgccgcctgccttgcccgctgctggacaggggctaggttgctgggcactgataa ttccgtggtgttgtcggggaagggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccct ttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaag aaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatct gagcctgggagctctctggctaactagggaacctactgcttaagcctcaataaagcttgccttgagtgcttCAAGTAGTGTGT
GCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGT
GTGGAAAATCTCTAGCagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtg agaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagt tgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgccc agttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagta gtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaa cgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagagg cccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggt gtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcg ccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatta gggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgtt ccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctg atttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctattt gtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatagcacctagatcaagagacag gatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgg gcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgt ccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgac gttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgaga aagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcat cgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaa ctgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtg gaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatatt gctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcg ccttcttgacgagttcttctgaattattaacgcttacaatttcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatcag gtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgaccaaaatccctta acgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgctt gcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagca gagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctct gctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgc agcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtga gctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgca cgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctc gtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttcttt cctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcag cgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggc acgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttaca ctttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgc gcaattaaccctcactaaagggaacaaaagctggagctgcaagcttggccattgcatacgttgtatccatatcataatatgtacatttatat tggctcatgtccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatata tgg
SEQ ID NO:18 E9(slim)-HS3(slim)-E2(lst half of 1 and 5 slim)-HSlpro-mCit-
WPRE (Deleted 2nd half of Core Sub-Element 1 of Element 2 and Core-Sub Element 4 of Element 2)
Sequence from CMV (red arrow) going clockwise in Figure 21 AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGA
CCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGG
ACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAG
TACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAA
ATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGG
CAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGT
ACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCC
CATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAA
TGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGG
GAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGGTCTCTCTGGTTAGACCA
GATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAA
TAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGG
TAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCagtggcgcc cgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagag gcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtat taagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatg ggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctaca accatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaa gacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcaga cctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcaccca ccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcagga agcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctga gggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagat acctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagta ataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaa ttgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataa caaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaataga gttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaaga aggtggagagagagaeagagaeagateeattegattagtgaaeggatetegaeggtateggttaaettttaaaagaaaaggggggatt ggggggtaeagtgeaggggaaagaatagtagaeataatageaaeagaeataeaaaetaaagaattaeaaaaaeaaattaeaaaaatt caaaattttatcgatcacgagactagcctcgagCAAACCTTGTTATCTGAGAGAAAGCTAGAGCTTCC
TTCTTTCAGCCCCCAGAGACAATGTGGCCAGGCTCCGGAGGGCTGGGAAGATGA
GCAATGCTTGTGAGTCACCATGATAGGAAGCAGAAGGGTCAGGAAGTCCCTGGG
AGCAAGGCTTAGGGTTAGGGTTAGGGAGAAAACACTTGGGCCTGGAGGCTCGGG
GCAGGCTTCCTAGAGGGGAGGGGTACTAAAAGCCACCAAAACCAAGACAGCAAT
GAAAGTAACCTCTGGTCTTCCTAACTGCTCATTATACACTAATTATGATGCACTA
GCATGCTAAGAGACACTTCCACCAGCGCCATGACAGTGCCATGGCAACATCAAG
AAGTCACCCCACATGGTCTAAAAAGGGGAAGAACCTTCAGTTCCCGGAATTGCC
CACCCCTTTCCTGGAAAACTCATGAATAATCCACCCCTTGTTTAGCATATAATGA
CCTGAAGGGTAAGGGGGTGTGGAGGTTGTGAAGGCGGGAAGGGGGGTAGCCCCT
TCACCAATGTAAACAAGGATGTGGGTTCTGCGGCCACACTCTCCCCCGCCCTCCC
CAGCGGCATTTCCAGCAAGTCACATGTCCTGCGCACAGGCTGGGGGCCCCCTGCT
GCTGCCTTTCTTAGAAGCCAGGACCACAGAGCCCGCACAGTGAGCTACTTGGGG
AGCTATTTCTGTAGACTGAGCTTGGGGTACTGGGAGGCAGTGCTGTTGGCCTCCA
ACTCTCTGCTCCACGTTCCTTCCACTATTCAACACATGGCAGGAAGTCAGCCTGT
AGGGCTTCACACTACAGTCTAAGCTGACTGCCATCTGTCCCCATCCAGGCGAGGC
TGGAAACGGGGGCAGCAAAAAGCCCAGGGACAAAGTCCCCTTCCCAGGCACATG
TATTATGCACTCTGCACCAAGGAAACCTCCAGATAAGGTCTAGAGACCAAGGGC
CATGTACTTCGGGGGAGAGGCCAGAGGACTTCTGAGGTTTTACAGAGAAAAGCC
AAAGGCAGCCCAGTCAGGGGAAATGTGTAGCCATAGTGCCGATAAGGAAAGGCC
TTCAACCTGCCCTGGTCAGCTCTTCCTGTAAGTAGAGGCCCCTTACCGAAGGCCC
CAGTGGGAGGAAGGGTCGGGAGTActcgagTCAGCCTCAGGCTACCTAGGTGCTTT AGAAAGGAGGCCACCCAGGCCCATGACTACTCCTTGCCACAGGGAGCCCTGCAC
ACAGATGTGCTAAGCTCTCGCTGCCAGCCAGAGGGAGGAGGGTCTGAGCCAGTC
AGAAGGAGATGGGCCCCAGAGAGTAAGAAAGGGGGAGGAGGACCCAAGCTGAT
CCAAAAGGTGGGTCTAAGCAGTCAAGTGGAGGAGGGTTCCAATCTGATGGCGGA
GGGCCCAAGCTCAGCCTAACGAGGAGGCCAGGCCCACCAAGGGGCCCCTGGAGG
ACTTGTTTCCCTTGTCCCTTGTGGTTTTTTGCATTTCCTGTTCCCTTGCTGCTCATT
GCGGAAGTTCCTCTTCTTACCCTGCACCCAGAGCCTCGCCAGAGAAGACAAGGG
CAGAAAGCACCATGgtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgac gtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccac cggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgatgtgcttcgcccgctaccccgaccacatgaa gcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagac ccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatc etggggeaeaagetggagtaeaaetaeaaeageeaeaaegtetatateatggeegaeaageagaagaaeggeateaaggtgaaett caagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgt gctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctgg agttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtgactgcaggaattcgagcatcttaccgccatttattcc catatttgttctgtttttcttgatttgggtatacatttaaatgttaataaaacaaaatggtggggcaatcatttacatttttagggatatgtaattac tagttcaggtgtattgccacaagacaaacatgttaagaaactttcccgttatttacgctctgttcctgttaatcaacctctggattacaaaattt gtgaaagattgactgatattcttaactatgttgctccttttacgctgtgtggatatgctgctttaatgcctctgtatcatgctattgcttcccgta cggctttcgttttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtccgtcaacgtggcgtggtgtgctct gtgtttgctgacgcaacccccactggctggggcattgccaccacctgtcaactcctttctgggactttcgctttccccctcccgatcgcca cggcagaactcatcgccgcctgccttgcccgctgctggacaggggctaggttgctgggcactgataattccgtggtgttgtcggggaa gggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctg gaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaag ggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctgg ctaactagggaacctactgcttaagcctcaataaagcttgccttgagtgcttCAAGTAGTGTGTGCCCGTCTGTTG
TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCT
CTAGCagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgc agcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactca tcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgc cccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttgga ggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaac cctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttc ccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagc gtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagc tctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagt gggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacact caaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacg cgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacatt caaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatagcacctagatcaagagacaggatgaggatcgtttcgca tgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcgg ctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaac tgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcggga agggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctga tgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcg gatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaagg cgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctgg attcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggc gaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctg aattattaacgcttacaatttcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatcaggtggcacttttcggggaaa tgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgaccaaaatcccttaacgtgagttttcgttccactg agcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccg ctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatact gttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggc tgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggg gggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgc ttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggggga aacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatg gaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtg gataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagc ggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaa agegggeagtgagegeaaegeaattaatgtgagttageteaeteattaggeaeeeeaggetttaeaetttatgetteeggetegtatgttg tgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaaggg aacaaaagctggagctgcaagcttggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgc catgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatgg
SEO ID NO:19 CMV:
AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGC CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG TCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTAC ATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT GGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTC CAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTT CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGA GGTCTATATAAGCAGAGCTCGTTTAGTGAACCG
SEO ID NO:20 5’R/U5:
GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACT
GCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG
ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGC
SEO ID NO:21 PSI:
Tcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtga gtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagt attaagcgggggag
SEO ID NO:22 RRE:
Tccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggta caggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattga ggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcc tggctgtggaaagatacct
SEO ID NO:23 WPRE:
Cccatatttgttctgtttttcttgatttgggtatacatttaaatgttaataaaacaaaatgg tggggcaatcatttacatttttagggatatgtaattactagttcaggtgtattgccacaaga caaacatgttaagaaactttcccgttatttacgctctgttcctgttaatcaacctctggatt acaaaatttgtgaaagattgactgatattcttaactatgttgctccttttacgctgtgtgga tatgctgctttaatgcctctgtatcatgctattgcttcccgtacggctttcgttttctcctc cttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtccgtcaacgtg gcgtggtgtgctctgtgtttgctgacgcaacccccactggctggggcattgccaccacctgt caactcctttctgggactttcgctttccccctcccgatcgccacggcagaactcatcgccgc ctgccttgcccgctgctggacaggggctaggttgctgggcactgataattccgtggtgttgt cggggaagggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacg agtcggatctccctttgggccgcctccccgcctgga
SEQ ID NO:24 3’ PPT: tttttaaaagaaaaggggggac
SEO ID NO:25 3’ delta U3/R/U5 tggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctc tggttagaccagatctgagcctgggagctctctggctaactagggaacctactgcttaagcc tcaataaagcttgccttgagtgcttCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTA
ACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGC
SEO ID NO:26 SV40 ori:
Atcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttt tatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggct tttttggaggcctagg
SEO ID NO:27 KANr:
Attgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggcta tgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcagg ggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgag gcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgt cactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcat ctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacg cttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtac tcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgc cagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacc catggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcga ctgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattg ctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctccc gattcgcagcgcatcgccttctatcgccttcttgacgagttcttctga
SEO ID NO:28 COLEI: agatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaa aaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaa ggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttag gccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttacca gtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttacc ggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaa cgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaa gggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgaggga gcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttg agcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcg SEO ID NO:29 Full sequence of WASVec lentiviral vector transfer plasmid with codon optimized WASp open reading frame: (sequence is beginning from the CMV):
AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGC CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG TCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTAC ATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT GGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTC CAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTT CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGA GGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGGTCTCTCTGGTTAGACCAGATCTGAGC CTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAG TGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCC TTTTAGTCAGTGTGGAAAATCTCTAGCagtggcgcccgaacagggacttgaaagcgaaaggg aaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcga ggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatg ggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggtta aggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctaga acgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggac agctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagca accctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagat agaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacct ggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaat tgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagag cagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgca gcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaa caatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatca agcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctgggg atttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggag taataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaatta acaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaat gaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaa ttggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatag tttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcag acccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggaga gagagacagagacagatccattcgattagtgaacggatctcgacggtatcggttaactttta aaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaaca gacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgatcacga gactagcctcgagCAAACCTTGTTATCTGAGAGAAAGCTAGAGCTTCCTTCTTTCAGCCCCC AGAGACAATGTGGCCAGGCTCCGGAGGGCTGGGAAGATGAGCAATGCTTGTGAGTCACCATG ATAGGAAGCAGAAGGGTCAGGAAGTCCCTGGGAGCAAGGCTTAGGGTTAGGGTTAGGGAGAA AACACTTGGGCCTGGAGGCTCGGGGCAGGCTTCCTAGAGGGGAGGGGTACTAAAAGCCACCA AAACCAAGACAGCAATGAAAGTAACCTCTGGTCTTCCTAACTGCTCATTATACACTAATTAT GATGCACTAGCATGCTAAGAGACACTTCCACCAGCGCCATGACAGTGCCATGGCAACATCAA GAAGTCACCCCACATGGTCTAAAAAGGGGAAGAACCTTCAGTTCCCGGAATTGCCCACCCCT TTCCTGGAAAACTCATGAATAATCCACCCCTTGTTTAGCATATAATGACCTGAAGGGTAAGG GGGTGTGGAGGTTGTGAAGGCGGGAAGGGGGGTAGCCCCTTCACCAATGTAAACAAGGATGT GGGTTCTGCGGCCACACTCTCCCCCGCCCTCCCCAGCGGCATTTCCAGCAAGTCACATGTCC TGCGCACAGGCTGGGGGCCCCCTGCTGCTGCCTTTCTTAGAAGCCAGGACCACAGAGCCCGC ACAGTGAGCTACTTGGGGAGCTATTTCTGTAGACTGAGCTTGGGGTACTGGGAGGCAGTGCT GTTGGCGATCTGGGGGAAAACCCCCTGAGGGTCCCATTTCTCTGCCACTGACCCCTTTGCCT GGCAGAAAGGCTCTGCTGCCTCCTAGCTGTGTAGCAATGGATAAGGCCTTTAACCTCTCTAA GCTGAAATGTCCTCATGCATAAGGTGGAGACAGTAACTCCTCTTCCATCTGCTGGGTGTTGT CAAAACTACACAGGAGGTTTGCCAAGCACCTAAGGCACAGTAGGTGGTCAAGAAACAGGAAC ACAATTTGTCATGACGACAATGATTCCTTCATATGCTAGTTACCCACGAGTCAGCCAGAATG TTGCCATAACCACTTATGAAGCCCTTCCTCATTTCTGTTGCAATTCCTCAGCCAGAGGAAAG CCCCACACAGCCCTTGCCACTCCCCCATGGCCCATGTAGAAACAACCATGTGGCCTTCACTC TGCCCACAGAAGCTAGCACCAGGACCCTGGTCAGGGTTAGAGGTTTCTGCTGAGTCAAAGCC ACATGGAGGGAGGGAGCAAGGGAGAGATGCAGAGTCATGTTTCCAGGAGGAGGTTATCTGAG CATAACAGGGACAGGGTGGGCCACAGGATACCTCTGAGGCTCAGGTTCCCACCTCCACTCCA CCCTCCAACTCTCTGCTCCACGTTCCTTCCACTATTCAACACATGGCAGGAAGTCAGCCTGT AGGGCTTCACACTACAGTCTAAGCTGACTGCCATCTGTCCCCATCCAGGCGAGGCTGGAAAC GGGGGCAGCAAAAAGCCCAGGGACAAAGTCCCCTTCCCAGGCACATGTATTATGCACTCTGC ACCAAGGAAACCTCCAGATAAGGTCTAGAGACCAAGGGCCATGTACTTCGGGGGAGAGGCCA GAGGACTTCTGAGGTTTTACAGAGAAAAGCCAAAGGCAGCCCAGTCAGGGGAAATGTGTAGC CATAGTGCCGATAAGGAAAGGCCTTCAACCTGCCCTGGTCAGCTCTTCCTGTAAGTAGAGGC CCCTTACCGAAGGCCCCAGTGGGAGGAAGGGTCGGGAGTActcgagTCAGCCTCAGGCTACC TAGGTGCTTTAGAAAGGAGGCCACCCAGGCCCATGACTACTCCTTGCCACAGGGAGCCCTGC ACACAGATGTGCTAAGCTCTCGCTGCCAGCCAGAGGGAGGAGGGTCTGAGCCAGTCAGAAGG AGATGGGCCCCAGAGAGTAAGAAAGGGGGAGGAGGACCCAAGCTGATCCAAAAGGTGGGTCT AAGCAGTCAAGTGGAGGAGGGTTCCAATCTGATGGCGGAGGGCCCAAGCTCAGCCTAACGAG GAGGCCAGGCCCACCAAGGGGCCCCTGGAGGACTTGTTTCCCTTGTCCCTTGTGGTTTTTTG CATTTCCTGTTCCCTTGCTGCTCATTGCGGAAGTTCCTCTTCTTACCCTGCACCCAGAGCCT CGCCAGAGAAGACAAGGGCAGAAAGCACCATGTCTGGCGGACCTATGGGAGGTAGACCTGGT GGAAGAGGTGCCCCCGCCGTGCAGCAGAACATCCCCAGCACCCTGCTGCAGGACCACGAGAA CCAGCGCCTGTTCGAGATGCTGGGCCGCAAGTGCCTGACCCTGGCCACCGCCGTGGTGCAGC TGTACCTGGCCCTGCCCCCCGGCGCCGAGCACTGGACCAAGGAGCACTGCGGCGCCGTGTGC TTCGTGAAGGACAACCCCCAGAAGAGCTACTTCATCCGCCTGTACGGCCTGCAGGCCGGCCG CCTGCTGTGGGAGCAGGAGCTGTACAGCCAGCTGGTGTACAGCACCCCCACCCCCTTCTTCC ACACCTTCGCCGGCGACGACTGCCAGGCCGGCCTGAACTTCGCCGACGAGGACGAGGCCCAG GCCTTCCGCGCCCTGGTGCAGGAGAAGATCCAGAAGCGCAACCAGCGCCAGAGCGGCGACAG AAGGCAACTGCCTCCTCCACCTACACCAGCCAACGAGGAAAGAAGAGGCGGACTGCCCCCCC TGCCTCTTCATCCTGGCGGAGATCAAGGTGGACCTCCTGTGGGACCACTGTCTCTTGGCCTG GCCACCGTGGACATCCAGAACCCCGACATCACCAGCAGCCGCTACCGCGGCCTGCCCGCCCC CGGCCCCAGCCCCGCCGACAAGAAGCGCAGCGGCAAGAAGAAGATCAGCAAGGCCGACATCG GCGCCCCCAGCGGCTTCAAGCACGTGAGCCACGTGGGCTGGGACCCCCAGAACGGCTTCGAC GTGAACAACCTGGACCCCGACCTGCGCAGCCTGTTCAGCCGCGCCGGCATCAGCGAGGCCCA GCTGACCGACGCCGAGACCAGCAAGCTGATCTACGACTTCATCGAGGACCAGGGCGGCCTGG AGGCCGTGCGCCAGGAGATGCGCCGCCAGGAGCCCCTGCCACCACCTCCACCTCCATCTAGA GGCGGCAACCAGCTGCCCCGCCCTCCTATCGTTGGCGGCAACAAGGGAAGATCTGGCCCTCT GCCTCCTGTGCCTCTGGGAATCGCTCCACCACCACCAACACCTAGAGGCCCGCCTCCACCAG GCAGAGGTGGTCCTCCGCCGCCACCTCCTCCAGCCACCGGCCGCAGCGGCCCCCTTCCTCCT CCACCACCTGGTGCTGGTGGACCTCCAATGCCACCGCCACCGCCTCCGCCACCTCCGCCTCC AAGTTCTGGAAATGGACCTGCTCCTCCTCCTTTGCCTCCTGCTTTGGTTCCTGCTGGCGGAT TGGCTCCAGGCGGAGGAAGAGGCGCACTGCTGGACCAGATCCGCCAGGGCATCCAGCTGAAC AAGACCCCTGGCGCTCCTGAGAGTTCTGCTCTGCAACCGCCACCACAGTCTAGCGAAGGACT TGTGGGAGCCCTGATGCACGTGATGCAGAAGCGCAGCCGCGCCATCCACAGCAGCGACGAGG GCGAGGACCAGGCCGGCGACGAGGACGAGGACGACGAGTGGGACGACTAActgcaggaattc gagcatcttaccgccatttattcccatatttgttctgtttttcttgatttgggtatacattt aaatgttaataaaacaaaatggtggggcaatcatttacatttttagggatatgtaattacta gttcaggtgtattgccacaagacaaacatgttaagaaactttcccgttatttacgctctgtt cctgttaatcaacctctggattacaaaatttgtgaaagattgactgatattcttaactatgt tgctccttttacgctgtgtggatatgctgctttaatgcctctgtatcatgctattgcttccc gtacggctttcgttttctcctccttgtataaatcctggttgctgtctctttatgaggagttg tggcccgttgtccgtcaacgtggcgtggtgtgctctgtgtttgctgacgcaacccccactgg ctggggcattgccaccacctgtcaactcctttctgggactttcgctttccccctcccgatcg ccacggcagaactcatcgccgcctgccttgcccgctgctggacaggggctaggttgctgggc actgataattccgtggtgttgtcggggaagggcctgctgccggctctgcggcctcttccgcg tcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattc gagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaa aagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgct tgtactgggtctctctggttagaccagatctgagectgggagctctctggctaactagggaa cctactgcttaagcctcaataaagcttgccttgagtgcttCAAGTAGTGTGTGCCCGTCTGT TGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGC agtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcaga gagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaa atttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaat gtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaact ccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggc cgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctag ggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttac aacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccct ttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcag cctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggtta cgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttccct tcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagg gttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcac gtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttcttt aatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttga tttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaat ttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatg tgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgaga caataaccctgataaatgcttcaataatagcacctagatcaagagacaggatgaggatcgtt tcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctat tcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtca gcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgca agacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcg acgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctc ctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggct gcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgag cacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcagggg ctcgcgccagccgaactgttcgccaggctcaa ggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttct ggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcg gcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttc tgaattattaacgcttacaatttcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatcaggtggcacttttcgggga aatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgaccaaaatcccttaacgtgagttttcgttcca ctgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacca ccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaat actgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagt ggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaac ggggggttegtgeaeaeageeeagettggagegaaegaeetaeaeegaaetgagataeetaeagegtgagetatgagaaagegee acgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagg gggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagc ctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgatt ctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgag gaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgac tggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgt atgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaa agggaacaaaagctggagctgcaagcttggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacatt accgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatgg

Claims

CLAIMS What is claimed is:
1. A recombinant lenti viral vector (LV) for the treatment of Wiskott- Aldrich Syndrome (WAS), said vector comprising: an expression cassette comprising: a nucleic acid encoding an effective fragment of the endogenous promoter of the WAS gene where said promoter has maximum length of 600 bp and contains the sequence of HSlpro (SEQ ID NO:l); and a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp) operably linked to said effective fragment of the endogenous promoter of the WAS gene.
2. The vector of claim 1, wherein the sequence of said effective fragment of the endogenous promoter of the WAS gene consists of the sequence of HSlpro (SEQ ID NO:l).
3. The vector according to any one of claims 1-2, wherein said expression cassette comprises a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8) or an effective fragment thereof.
4. The vector of claim 3, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).
5. The vector of claim 4, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment consists of enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).
6. The vector of claim 3, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of the first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).
7. The vector of claim 6, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment consists of the first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub element 5 (SEQ ID NO: 8).
8. The vector of claim 3, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of the 1st half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:3).
9. The vector according to any one of claims 3 and 8, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of the second half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:4).
10. The vector according to any one of claims 3 and 8-9, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core Sub-Element 2 of Enhancer Element 2 (SEQ ID NO:5).
11. The vector according to any one of claims 3 and 8-10, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core Sub-Element 3 of Enhancer Element 2 (SEQ ID NO:6).
12. The vector according to any one of claims 3 and 8-11, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core-Sub Element 4 of Enhancer Element 2 (SEQ ID
NO:7).
13. The vector according to any one of claims 3 and 8-12, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core-Sub Element 5 of Enhancer Element 2 (SEQ ID NO:8).
14. The vector according to any one of claims 1-13, wherein said expression cassette comprises enhancer element HS3 (SEQ ID NO: 9) or an effective fragment thereof.
15. The vector of claim 14, wherein said expression cassette comprises an effective fragment of enhancer element HS3 wherein said fragment comprises or consists of HS3 core sequence (SEQ ID NO: 10).
16. The vector of claim 15, wherein said expression cassette comprises an effective fragment of enhancer element HS3 wherein said fragment consists of HS3 core sequence (SEQ ID NO: 10).
17. The vector according to any one of claims 1-16, wherein said expression cassette comprises enhancer element E9 (SEQ ID NO: 11) or an effective fragment thereof.
18. The vector of claim 17, wherein said expression cassette comprises an effective fragment of enhancer element E9 wherein said fragment comprises or consists of enhancer element E9 core sequence (SEQ ID NO: 12).
19. The vector of claim 18, wherein said expression cassette comprises an effective fragment of enhancer element E9 wherein said fragment consists of enhancer element E9 core sequence (SEQ ID NO: 12).
20. The vector according to any one of claims 1-2, wherein said expression cassette comprises: a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8); and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HSlpro (SEQ ID NO:l).
21. The vector of claim 20, wherein said vector comprises the features shown in Figure 18 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
22. The vector of claim 20, wherein said vector comprises the sequence show in SEQ ID NO: 15 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp)..
23. The vector according to any one of claims 1-2, wherein said expression cassette comprises: enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO: 12); enhancer element HS3 sequence comprising or consisting of HS3 core sequence (SEQ ID NO: 10); a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8); and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HSlpro (SEQ ID NO:l).
24. The vector of claim 23, wherein said vector comprises the features shown in Figure 19 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
25. The vector of claim 23, wherein said vector comprises the sequence show in SEQ ID NO: 16 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
26. The vector of claim 25, wherein said vector comprises the sequence show in SEQ ID NO:29.
27. The vector according to any one of claims 1-2, wherein said expression cassette comprises:! enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO: 12); enhancer element HS3 sequence comprising or consisting of HS3 core sequence (SEQ ID NO: 10); enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8); and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HSlpro (SEQ ID NO:l).
28. The vector of claim 27, wherein said vector comprises the features shown in Figure 20 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
29. The vector of claim 27, wherein said vector comprises the sequence show in SEQ ID NO: 17 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
30. The vector according to any one of claims 1-2, wherein said expression cassette comprises: enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO: 12); enhancer element HS3 sequence comprising or consisting of HS3 core sequence (SEQ ID NO: 10); a first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8); and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HSlpro (SEQ ID NO:l).
31. The vector of claim 30, wherein said vector comprises the features shown in Figure 21 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
32. The vector of claim 30, wherein said vector comprises the sequence show in SEQ ID NO: 18 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).
33. The vector according to any one of claims 1-32, wherein said nucleic acid that encodes a nucleic acid that encodes WASp protein is a WAS cDNA or a codon- optimized WAS gene.
34. The vector of claim 33, wherein said nucleic acid that encodes a nucleic acid that encodes WASp protein is a WAS cDNA (SEQ ID NO: 13).
35. The vector of claim 33, wherein said nucleic acid that encodes a nucleic acid that encodes WASp protein is a codon optimized WAS.
36. The vector of claim 35, wherein the sequence of said nucleic acid that encodes WASP is a codon optimized WAS selected from the group consisting of jCAT codon optimized WAS, GeneArt optimized WAS, and IDT optimized WAS.
37. The vector according to any one of claims 1-36, wherein said vector comprises a y region vector genome packaging signal.
38. The vector according to any one of claims 1-37, wherein said vector comprise a 5' LTR comprising a CMV enhancer/promoter.
39. The vector according to any one of claims 1-38, wherein said vector comprises a Rev Responsive Element (RRE).
40. The vector according to any one of claims 1-39, wherein said vector comprises a central polypurine tract.
41. The vector according to any one of claims 1-40, wherein said vector comprises a post-translational regulatory element.
42. The vector of claim 41, wherein the posttranscriptional regulatory element is modified Woodchuck Post-transcriptional Regulatory Element (WPRE).
43. The vector according to any one of claims 1-42, wherein said vector is incapable of reconstituting a wild-type lentivirus through recombination.
44. The vector according to any one of claims 1-43, wherein said vector shows high expression in megakaryocytes.
45. The vector according to any one of claims 1-44, wherein said vector restores T, B and NK cell counts and function when administered to a mammal having WAS.
46. A host cell transduced with a vector according to any one of claims 1- 45.
47. The host cell of claim 46, wherein the cell is a stem cell.
48. The host cell of claim 47, wherein said cell is a stem cell derived from bone marrow, and/or from umbilical cord blood, and/or from peripheral blood.
49. The host cell of claim 46, wherein the cell is a human hematopoietic progenitor cell.
50. The host cell of claim 49, wherein the human hematopoietic progenitor cell is a CD34+ cell.
51. A method of treating Wiskott-Aldrich Syndrome (WAS), in a subject, said method comprising: transducing a stem cell and/or progenitor cell from said subject with a vector according to any one of claims 1-45; and transplanting said transduced cell or cells derived therefrom into said subject where said cells or derivatives therefrom express said WASp protein.
52. The method of claim 51 , wherein the cell is a stem cell.
53. The host cell of claim 51, wherein said cell is a stem cell derived from bone marrow.
54. The method of claim 51 , wherein the cell is a human hematopoietic stem and progenitor cell.
55. The method of claim 54, wherein the human hematopoietic progenitor cell is a CD34+ cell.
56. A recombinant nucleic acid comprising one or more of the following: a nucleic acid sequence comprising or consisting of a minimal endogenous promoter of the WAS gene said minimal endogenous promoter comprising or consisting of HSlpro (SEQ ID NO:l); and/or a nucleic acid sequence comprising or consisting of a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8) or an effective fragment thereof; a nucleic acid sequence comprising or consisting of a 1st half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO: 3) ; and/or a nucleic acid sequence comprising or consisting of a 2nd half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:4); and/or a nucleic acid sequence comprising or consisting of a Core Sub- Element 2 of Enhancer Element 2 (SEQ ID NO: 5); and/or a nucleic acid sequence comprising or consisting of a Core Sub- Element 3 of Enhancer Element 2 (SEQ ID NO: 6); and/or a nucleic acid sequence comprising or consisting of a Core-Sub Element 4 of Enhancer Element 2 (SEQ ID NO:7); and/or a nucleic acid sequence comprising or consisting of a Core-Sub Element 5 of Enhancer Element 2 (SEQ ID NO: 8); and/or a nucleic acid sequence comprising or consisting of enhancer element HS3 (full) (SEQ ID NO:9) or an effective fragment thereof; and/or a nucleic acid sequence comprising or consisting of Enhancer element HS3 core (SEQ ID NO: 10); and/or a nucleic acid sequence comprising or consisting of Enhancer element E9 (full) (SEQ ID NO: 11) or an effective fragment thereof; and/or a nucleic acid sequence comprising or consisting of Enhancer element E9 core (SEQ ID NO: 12).
57. The nucleic acid of claim 56, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a minimal endogenous promoter of the WAS gene said minimal endogenous promoter comprising or consisting of HSlpro (SEQ ID NO:l).
58. The nucleic acid of claim 56, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8) or an effective fragment thereof.
59. The nucleic acid of claim 56, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a 1st half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO: 3).
60. The nucleic acid according to any one of claims 56, and 59, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a 2nd half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:4).
61. The nucleic acid according to any one of claims 56, and 59-60, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core Sub-Element 2 of Enhancer Element 2 (SEQ ID NO:5).
62. The nucleic acid according to any one of claims 56, and 59-61, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core Sub-Element 3 of Enhancer Element 2 (SEQ ID NO:6).
63. The nucleic acid according to any one of claims 56, and 59-62, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core-Sub Element 4 of Enhancer Element 2 (SEQ ID NO:7).
64. The nucleic acid according to any one of claims 56, and 59-63, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core-Sub Element 5 of Enhancer Element 2 (SEQ ID NO: 8).
65. The nucleic acid according to any one of claims 56, and 59-64, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of enhancer element HS3 (full) (SEQ ID NO:9) or an effective fragment thereof.
66. The nucleic acid according to any one of claims 56, and 59-65 , wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of Enhancer element HS3 core (SEQ ID NO: 10).
67. The nucleic acid according to any one of claims 56, and 59-66, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of Enhancer element E9 (full) (SEQ ID NO: 11) or an effective fragment thereof.
68. The nucleic acid according to any one of claims 56, and 59-67, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of Enhancer element E9 core (SEQ ID NO: 12).
69. The nucleic acid according to any one of claims 56-68, wherein said nucleic acid comprises an expression cassette.
70. The nucleic acid of claim 69, wherein said expression cassette is effective to express WASp when transduced into a mammalian cell.
71. The nucleic acid of claim 56, wherein said nucleic acid comprises a vector according to any one of claims 1-45.
EP20886855.4A 2019-11-11 2020-11-10 Lentiviral vectors in hematopoietic stem cells to treat wiskott-aldrich syndrome (was) Pending EP4058587A4 (en)

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